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DNA Fragment Analysis by Capillary Electrophoresis User Guide

1. Peak Amplitude Threshold of 2131228 mmm PNG NEN PAA ANA 22222 haa aa be dla aa Nh i W i UN MW N i JM ua oT UU MP n H HP NTE LH BAPI LEN L WO Ny Poor or non specific See PCR troubleshooting on page 176 amplification See Non specific amplification on page 32 for more information PCR inhibition See PCR troubleshooting on page 176 Sample was prepared with Prepare the sample with Hi Di Formamide water instead of Hi Di Formamide Degraded or incorrectly stored Use fresh properly stored Hi Di Formamide See Hi Di Formamide Hi Di Formamide storage on page 82 Air bubble at bottom of sample Centrifuge the plate before running tube 164 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible cause Action No signal or low signal High salt concentration Salt preferentially injects smaller fragments and continued inhibits injection of larger fragments so the majority of salt may have been injected in the first injection Re inject the sample If signal intensity does not increase see Desalting on page 190 Injection time too low See Optimizing electrokinetic injection parameters on page 78 Sample concentration too low See Optimizing sample loading concentration on page 76 Sample volume too low e Sample volume must be 210 uL for 3500 Series 373
2. Multiple binding sites Primers do not bind effectively to target sequences with known secondary structures For example hairpin structures are often found in regions of high G C content or in RNA sequences If you must design primers to a specific target region with the potential for hairpin formation you may try addition of DMSO to your reaction or other commercially available kits for difficult template amplification Amplification of the desired target sequence requires minimizing primer binding to secondary sites in the DNA and to other primers Note This applies to template genomic DNA The probability of binding to secondary sites is lower for low complexity templates such as plasmid DNA Ideally the binding of the primer to the desired template region e Is strongest at the 5 end Generally requires a higher more negative value to maximize base pairing energy than 9 8 kcal mole at the 3 end see Table 4 on page 30 As a general rule binding at the 3 end should be weaker than 9 8 kcal mole When alleles differ in size by ten or more base pairs you may observe preferential amplification of shorter PCR products over longer ones Walsh et al 1992 This will also occur when amplifying low copy number DNA or DNA isolated from paraffin embedded tissues Figure 3 on page 32 is an example of preferential amplification of the D55346 marker In both the normal top panel and tu
3. Sample File Panel Dye Allele 1 Allele 2 Allele 3 Allele4 Allele5 Allele6 Allele7 Allele Allele9 Allele 10 Allele 11 Allele 12 Allele 13 Allele 14 Allele 15 Allele 1 1 AFLP corn F1 AFLP TutorialPan B 0 1 0 1 0 0 1 1 0 1 1 0 2 AFLP corn P1 AFLP Tutorial Pan B 1 1 0 0 1 0 0 1 1 0 1 0 0 0 1 1 3 AFLP com P2 AFLP TutorialPanB 1 0 1 1 1 1 1 0 1 1 0 0 1 1 0 0 ENS i For more For documents and publications see AFLP applications on page 200 information For ordering information see Ordering Information on page 193 130 DNA Fragment Analysis by Capillary Electrophoresis Chapter 8 Fingerprinting 8 Terminal restriction fragment length polymorphism T RFLP Overview Terminal restriction fragment length polymorphism T RFLP analysis is a mapping technique used to study complex microbial communities based on variation in the 165 rRNA gene Osborn and Moore et al It is culture independent rapid sensitive and reproducible and does not require genomic sequence information Principle of the In T RFLP analysis fluorescently labeled DNA is digested with restriction enzymes analysis that have 4 base pair recognition sites This step generates fluorescently labeled terminal restriction fragments The fragments in the digest are then separated by capillary electrophoresis Profiles can then be compared between samples or matched to a
4. 0 0 0 e cece cece eee ees 62 AmplilagGold 1 eb te eae on wae ea eer RR a RKE KARE L RA 62 General PER ee riae boda die beh ee HYG e Hic e DIR RE E PER 62 tS SY eee m trien E uS M bui A 62 Timedelsase PCR tint Ane tad Lot teh do Serre nt tef ea darte Anal coed 62 Touchdown PUR enr bre DET bones beside terere 63 XEPCR cod Ense ERE dd RAT ebbe eei dex ttd Gee wee dice Mns 63 Optimizing thermal cycling parameters 0c cece eect ees 63 Optimizing temperatures me RE T 2 R Eee toe e vetet C i eg 63 Guidelines ocu iore ses lor eatis eame RU Vay eM IE E Ge aes 64 Avoiding contamination s eee nett eeeeeeeees 64 PCR setup work area isi bU UR Gaede eg dest RAE RAISES ARE EET 64 Amplified DNA work area ssssssssssslllsllls ss e eee eeeees 65 Avoiding contamination from the environment 0 000ecee cece cette ees 65 Avoiding PCR product carryover 000 c cece e eee eect eee eeees 65 Formore information i Re d tee eer eee aa e deu eee e i ep 66 DNA Fragment Analysis by Capillary Electrophoresis 5 Contents S CHAPTER 4 Optimizing Capillary Electrophoresis 67 Safety Information nea Sr uv IERI Kaa gested 1a Med eins Meneses ed eat E ERR 68 OVERVIEW o see cs Soe ard ZT K e eed Hed Ov 4g de pel peo R eee MiGs Pee eRe MOE pese 68 Life Technologies Genetic Analyzers 0c aee 68 Overview of run modules 000 cece cece n 69 Using conttrols uh er cnt Em eet e tee tia e pri Res 69 3500 Se
5. 191 DNA Fragment Analysis by Capillary Electrophoresis 151 Troubleshooting workflow Problems with data can be caused during any step of the experiment AS EE Sample reactions poe nung Instrument run Data analysis P assay workflows Instrumentrun Data collection Interpretation template and kit protocols setup When troubleshooting follow this workflow to identify the problem 1 Make sure you understand the basics of the experiment The chemistry Labeling of the samples How the genetic analyzer collects data How the data analysis software performs sizing and peak detection Review the experiment for errors in primer design sample quantitation and purification pipetting problems software preference settings and other common mistakes 2 Examine the data Evaluate the problem as specifically as possible e sita problem with the sample peaks the baseline or the peaks of only one color Look for patterns Does the problem exist in all parts of the run or does it affect only DNA fragments of a certain length in a specific capillary in a certain area of the plate multiple runs Is the problem visible in raw data analyzed data log files Continue to refine the description of the problem as specifically and thoroughly as possible 3 In general check first for the issues that can be resolved most easily Review Dataquality Analysis settings Data collection Experimental setup For
6. Applied Biosystems 3500xL Genetic Analyzer 3130 or 3130xl Genetic Analyzer 3730 or 3730xl DNA Analyzer 310 Genetic Analyzer 3500 3500xL Analyzer materials Anode Buffer Container ABC 4393927 Cathode Buffer Container CBC 4408256 Septa Cathode Buffer Container 3500 Series 4410715 Conditioning Reagent 4393718 Capillary Array 36 cm 8 Capillary 3500 Genetic Analyzers 4404683 Capillary Array 50 cm 8 Capillary 3500 Genetic Analyzers 4404685 Capillary Array 36 cm 24 Capillary 3500xL Genetic Analyzers 4404687 Capillary Array 50 cm 24 Capillary 3500xL Genetic Analyzers 4404689 GeneScan Size Standards See GeneScan size standards on page 197 Matrix Standard Kits See Matrix standards for spectral calibration on page 197 POP 49 Polymer 960 Samples 3500 3500xL Genetic Analyzers 4393710 POP 4 Polymer 384 Samples 3500 3500xL Genetic Analyzers 4393715 POP 6 Polymer 960 Samples 3500 3500xL Genetic Analyzers 4393712 POP 6 Polymer 384 Samples 3500 3500xL Genetic Analyzers 4393717 POP 7 Polymer 960 Samples 3500 3500xL Genetic Analyzers 4393710 POP 7 Polymer 384 Samples 3500 3500xL Genetic Analyzers 4393715 8 Tube Retainer and Base Set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 well Retainer and Base Set Standard 3500 3500xL Genetic An
7. Copy Number Variation CNV Aneuploidy detection Applications The applications in this guide are identified as one of the following described in this guide Category Description Life Technologies supported Life Technologies has tested and validated this protocol on the instrument system specified The technical support and field application specialists have been trained to support this protocol Life Technologies demonstrated Life Technologies has tested this protocol but has not validated for the instrument system specified Certain components of the protocol workflow such as reagent kits and other protocols for preparation of reagents may not be available through Life Technologies Supporting documentation such as application notes may be available from Life Technologies and or third parties Limited support is available from Life Technologies Customer demonstrated Life Technologies has not tested this protocol However at least one customer or third party has reported successfully performing this protocol on the instrument system specified Life Technologies cannot guarantee instrument and reagent performance specifications with the use of customer demonstrated protocols However supporting documentation from Life Technologies and or third parties may be available and Life Technologies may provide basic guidelines in connection with this protocol DNA Fragment Analysis by Capillary Electrophoresis
8. e NucAway Spin Columns NucAway Spin Columns remove unincorporated nucleotides and salts after probe synthesis reactions Rehydrate the column centrifuge to remove the interstitial fluid add the sample to the top of the column and centrifuge again Amicon Centricon 100 Microconcentrator or Centricon 30 for fragments smaller than 130 bp Ethanol precipitation of the pooled PCR product followed by resuspension in distilled deionized water Refer to Molecular cloning Sambrook Fritsch and Maniatis 1989 for protocols Sample dialysis on a filter membrane a Floata Millipore VS filter Millipore Part no VSWP 02500 shiny side up on top of 50 mL of deionized autoclaved water in a 50 mL conical plastic tube b Carefully spot 15 uL of sample on top of the filter using an appropriate pipette DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting c Dialyze the sample for 20 minutes d Using a pipette very carefully remove the sample and dilute Evaluating 310 Genetic Analyzer multicomponent matrix quality Purpose of the multicomponent matrix Factors affecting matrix quality When to create a new matrix Virtual Filter Set C Identifying matrix problems The multicomponent matrix compensates for the overlap of different dye colors by subtracting out in each dye s detection range the portion of the signal due to fluorescence from other dyes Reagent quality
9. 6 4 kcal Note Although a G C dinucleotide at the 3 end of the primer can stabilize the template primer binding complex when using thermostable enzymes such as AmpliTaq DNA Polymerase a 3 G C can also lead to false priming if you do not optimize PCR conditions Topal and Fresco 1976 Effects of primer secondary structure Strings of Gs and Cs can form internal non Watson Crick base pairs Sarocchi et al 1970 that disrupt stable primer annealing Although this anomalous behavior is difficult to predict a good general rule is to avoid runs of more than three consecutive Gs in primers DNA Fragment Analysis by Capillary Electrophoresis Effects of template secondary structure Selective amplification Preferential amplification Chapter 2 Experimental Design However a short run of Gs at or near the 5 end of a primer will not disrupt the stability of primer template complexes because 5 positioning does not lead to involvement in disruptive secondary structures for example primer dimer or duplex loops Similarly self complementary sequences within the primer can lead to the formation of hairpin structures that disrupt stable primer binding to template A stable hairpin can form with just four G C base pairs in the stem and three bases in the loop Summer et al 1985 Figure 2 Figure 2 Secondary structures in primers Hairpin loop Hairpin loop Forward and sequence sequence reverse primers eS L
10. Note Do not shake polymer or introduce bubbles DNA Fragment Analysis by Capillary Electrophoresis 159 Size standard lonic buffer strength not applicable to 3500 Series instruments Sizing quality issues can be caused by a degraded or improperly stored size standard A size standard can be degraded by using improperly stored Hi Di Formamide see Hi Di Formamide storage on page 82 Conductivity changes in the buffer affect the run current and can cause the following e Decreased sample resolution Slower than expected migration of size standard peaks Low current Possible causes of buffer issues Water impurities High salt concentration Expired or incorrectly stored buffer Instrument and ambient condition issues Capillary array Pump large bubbles 160 Degraded capillary arrays can cause Decreased sample resolution Broad lagging peaks Possible causes of degraded capillary array performance Capillary array life is exceeded Capillary array is left idle or dries out Poor quality DNA or water or degraded Hi Di formamide has introduced contaminants that ultimately affect the current flow through the capillaries Water wash is not performed as recommended or contaminated water is used for the wash Clogged capillary If the same capillary always fails run Hi Di Formamide blanks then an installation standard or size standard as controls through the capillary see Running co
11. Polynomial curve of degree 3 red 1000 Polynomial curve of degree 2 green 500 DNA Fragment Analysis by Capillary Electrophoresis 95 Effects of Increasing the Window Size Value 96 In the figure below both polynomial curves have a degree of 3 and the window size value was increased from 15 red to 31 black data points As the cubic polynomial is stretched to fit the data in the larger window size the polynomial curve becomes smoother Note that the structure of the smaller trailing peak is no longer detected as a distinct peak from the adjacent larger peak to the right 2000 1500 1000 Window size value of 31 black Window size value of 15 fed T 500 40 45 50 55 60 65 70 75 80 85 DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software GeneMapper Software peak start and end settings The Slope Threshold for Peak Start and Slope Threshold for Peak End parameters adjust the start and end points of a peak The values assigned to these parameters can be used to better position the start and end points of an asymmetrical peak or a poorly resolved shouldering peak to more accurately reflect the peak position and area In general from left to right the slope of a peak increases from the baseline up to the apex From the apex down to the baseline the slope decreases negatively until it returns to zero at the baseline se
12. 1800 900 o IM 2n 29ampleo ew7 z 7 formanmide 33808 o TAM 3R 35ampies 507 37 tormamide 408u5 GS500 ROX size standard 1180 uS Solvent Resolution Peak Height Water 1 19 1 0 01 2700 300 408 uS Formamide 27S 1 15 0 05 2960 30 Formamide 3601S 1 20 0 08 879 4 Formamide 1000S 1 20 0 06 290 14 MM 4 4Sampiewn97 4 formamide 118005 Polymer handling and characteristics Improperly stored Hi Di Formamide can cause a variety of electrophoresis problems For information see Hi Di formamide on page 159 IMPORTANT Do not leave polymer on the instrument more than seven days Polymer left on the instrument for more than seven days causes a loss of resolution Avoid actions that introduce bubbles or particles into the polymer Dust in the polymer can cause data spikes To minimize bubbles and particles in polymer Close the polymer cap during storage to minimize exposure of the polymer to air Clean the polymer delivery system with deionized water e Discard capillaries that are exposed to dust or are dried out Change the buffer and water and discard the waste daily or before each set of runs Table 17 Polymer characteristics Instrument Polymer characteristics POP 49 Polymer Less viscous fast runs POP 6 Polymer More viscous slow runs POP 7 Polymer Less viscous fast runs not for use on 310 instruments DNA Fragment Analysis by C
13. Fo xx C 7 Electroph i ST 1 ectrophoresis jl li Lu Sample 1 ALL Lin L1 TETE E cL ee ee 2 Sample 2 MLM pl yy L 4 j i Sample 3 ail t t 8 AFLP is possible because the abundant complexity in eukaryotic genomic DNA means that it is statistically likely that enough restriction fragments will be short enough to successfully produce PCR amplicons that yield a unique fingerprint profile The power of AFLP analysis derives from its ability to quickly generate large numbers of marker fragments for any organism without prior knowledge of the genomic sequence In addition AFLP analysis requires only small amounts of starting template and can be used for a variety of genomic DNA samples DNA Fragment Analysis by Capillary Electrophoresis Applications Instrument and consumable recommendations Experiment and primer design recommendations Chapter 8 Fingerprinting E Fingerprints or AFLP band patterns can be used for many purposes For example AFLP analysis is often used in plant research where fingerprints can be compared to determine the plant variety or to compare the similarities between different plant varieties Life Technologies provides kits for performing AFLP on microbes and plants and reagents that are useful for performing AFLP on other organisms Some additional applications for AFLP analysis include Molecular diversity studies Zhao et al 20
14. Iwahana H Adzuma K Takahashi Y Katashima R Yoshimoto K and Itakura M 1995 Multiple fluorescence based PCR SSCP analysis with postlabeling Genome Res 4 275 282 Johnson E L Zhang D Emche S D et al 1994 Construction of libraries enriched for sequence repeats and jumping clones and hybridization selection for region specific markers Proc Natl Acad Sci USA 91 88 92 Lai Y and Sun F The relationship between microsatellite slippage mutation rate and the number of repeat units 2003 Molecular Biology and Evolution 20 12 2123 2131 Leroy X J and Leon K 2000 A rapid method for detection of plant genomic instability using unanchored microsatellite primers Plant Mol Biol Rep 2000 18 283a 283g Luo M C et al High throughput fingerprinting of bacterial artificial chromosomes using the SNaPshot labeling kit and sizing of restriction fragments by capillary electrophoresis Genomics 82 2003 378 389 Molecular Cloning 1987 Second Edition Cold Spring Harbor Laboratory NY Cold Spring Harbor Laboratory Press Murray V Monachawin C and England P R 1993 The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR Nucleic Acids Research 21 2395 2398 Orita M Iwahana H Kanazawa H et al 1989 Inter and Intra specific Variation among Five Erythroxylum Taxa Assessed by AFLP Proc Natl Acad Sci USA 86 2766 2770 Osborn A M Moore R B and T
15. 0 00 00e0e 97 m How the GeneMapper Software performs sizing isle eese 98 E GeneMapper Software sizing methods 100 m Evaluating data duat 104 Overview This chapter contains general information for data evaluation For application specific information on data evaluation see the application chapters later in this guide GeneMapper Software analyzes the data collected on Life Technologies genetic analyzers to size and genotype DNA fragments You can also use the GeneMapper Software data to perform relative quantitation for more information see Chapter 9 Relative Fluorescence Quantitation RFQ on page 143 Peak Scanner Software can be used for preliminary sizing How the software Both GeneMapper Software and Peak Scanner Software perform analysis on processes data original fsa files generated by the Data Collection Software fsa files Y Baselining Peak detection Size matching Size curve Y Genotyping Quality Value determination DNA Fragment Analysis by Capillary Electrophoresis 89 Precise versus accurate sizing Relative sizing Guidelines for consistent sizing 90 When evaluating sizing consider two metrics e Precision reproducibility The measure of the ability to generate the same size consistently for a given fragment obtained under the same conditions e Accuracy The measure of the abi
16. Applications described in this dude eee 17 What is capillary electrophoresis nuunuu nunnana nauar akanaka a 18 Fragment analysis workflow 202ce cece eee eee eee eee eee ees 19 S CHAPTER2 Experimental Design c cece eee ee eee 21 Experimental design considerations 21 DNA polymerase enzymes ssssssssssssssssss eee eee n rn rn rr 22 OVErVIEW icsiceunisntiuedhrsertaso Chale Pk Satie Ca Siti dae A dnd dd 22 Derivatives of Tth DNA polymerase cece eee eee se 24 Enzyme characteristics otserri ne eg des AR pA REPE ce deed yee 24 Fluorescent labeling methods sekers yesen T cece eee eee eee eee EEREN PAARA RA RT 25 Singleplexing versus multiplexing n anann annn rnnr rnnr rnrn rnrn 26 Singleplexing s sid ER bean hte edie eles Mies ANE 26 Multiplexing cic esd ecg eye Pad S Ay ee X Gey EEE RRR RR Mec R Se te ed 26 Multiplexing pooling strategies 27 Multiplex design software ccc cece eect m AT ER 28 Multiplexing guidelines 4 rc tele ed E eb ee ee ee 28 Primer design guidelines cece eee eee ee eee eee re 29 Primer design criteria cece eee eee eee rn 29 Primerdesign software 5 2 sues bee ETT RI ee eel ele 29 Factors affecting T and primer annealing 00 c cece eee eee eee 30 Effects of template secondary structure 0c cece eee ees 31 Selective amplification R S TR a eect e 31 Preferential amplification ssssssssssssssss
17. Light smoothing slightly reduces peak height in the electropherogram It does not affect tabular data e Heavy is useful for data from slower runs that display broad peaks or to avoid the detection of sharp edges This selection may reduce peak size or eliminate narrow peaks in the electropherogram It does not affect tabular data The Baseline Window adjusts the baselines of all detected dye colors to the same level for an improved comparison of relative signal intensity and helps to eliminate noise from the baseline Ifthe Baseline Window value is too low the baseline approaches the peaks and the data display shorter peaks Ifthe Baseline Window value is too high the baseline is too low and the data display elevated and possibly not baseline resolved peaks The Min Peak Half Width setting specifies the smallest full width at half maximum for peak detection Use a low value if the data display narrow peaks If the value is high noise spikes are ignored Use the Polynomial Degree and the Peak Window Size settings to adjust the sensitivity of the peak detection You can adjust these parameters to detect a single base pair difference while minimizing the detection of shoulder effects or noise Sensitivity increases with larger polynomial degree values and smaller window size values Conversely sensitivity decreases with smaller polynomial degree values and larger window size values The peak detector calculates the first deri
18. Three Optimized Workflows for CpG Island Methylation Profiling 106AP24 01 Microsatellite applications GeneMapper Software Getting Started Guide Microsatellite Analysis 4403672 Fact sheet Microsatellite Analysis on the Applied Biosystems 3130 Series Genetic Analyzers 106MI61 01 Uniparental disomy UPD analysis of chromosome 15 CO18249 Relative fluorescence quantitation applications Aneuploidy Detection by QF PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer 106AP28 01 200 DNA Fragment Analysis by Capillary Electrophoresis Documentation and Support Publication Document number Relative Fluorescent Quantitation on Capillary Electrophoresis Systems Screening for Loss of Heterozygosity 106AP15 01 in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper Software V3 7 SNP applications GeneMapper Software Getting Started Guide SNaPshot Kit Analysis 4403618 User Bulletin Using the SNaPshot Multiplex System with the POP 7 Polymer on Applied Biosystems 4367258 3730 3730xl DNA Analyzers and 3130 3130 SNaPshot Multiplex Kit Quick Reference Card 4323975 Demonstration of SNP Genotyping Using a Single Nucleotide Extension Method and the 3500 Series Genetic C012863 Analyzer Using the SNaPshot Multiplex System with the POP 7 Polymer on Applied Biosystems 3730 3730xl 4367258 DNA Analyzers and 3130 3130xl Genetic Analyzers Obtain SDSs Safet
19. gt Green gt Yellow gt Red see Emission and absorption excitation wavelengths and relative intensities on page 38 e Use less intense dyes for PCR product with good recovery rate Select dyes with absorption maxima that are as far apart as possible to avoid overlap and for easier generation of matrix spectral calibration see Emission and absorption excitation wavelengths and relative intensities on page 38 Consider the relative dye intensities and sample concentration see Emission and absorption excitation wavelengths and relative intensities on page 38 Example selecting The following figure shows the marker range allele distribution and allele dyes frequencies for the alligator microsatellite locus Amiu 8 for samples taken from Florida South Georgia and Texas Louisiana Figure7 Allele distribution for alligator marker Amip 8 in two populations 124 to 156 bp gt S E R FLsGA Using a hypothetical set of 10 markers as an example e For each marker determine the expected allele distribution either from published literature or from empirical testing Determine the dyes that are appropriate for the range of each marker of interest DNA Fragment Analysis by Capillary Electrophoresis 39 40 e For this hypothetical set of markers you might select 6 FAM VIC PET NED dyes and LIZ dye for the size standard see the table below This group of dyes corresponds to the G
20. including the beginning apex and end of each peak are tick marked in the electropherogram Comparing data s Use the same GeneScan size standard labeled with the same dye for all samples in a single study Compare peak areas heights and sizes in nucleotide bases only if fragments are labeled with the same dye For more information see Precise versus accurate sizing on page 90 and Relative sizing on page 90 Compare only data that is collected under the same conditions capillary array length polymer type and electrophoretic run conditions for the same study because these conditions affect the relative size of the fragment DNA Fragment Analysis by Capillary Electrophoresis 105 106 DNA Fragment Analysis by Capillary Electrophoresis Microsatellite Analysis B Overview of microsatellite analysis 6 60 c cee nes 107 B Applications screenie hene e ee tenet eee bte a vena vt 110 BW Instrument and consumable recommendations cece eee 111 E Experiment and primer design recommendations 000 0 000s 111 Maio A IIT 112 R Dataanalysi sete Ree ER RC REC UEE CHE REV C ER RR eene eh 112 B Common problems with microsatellite analysis 00 eee eae 113 S Identifying stutter products in microsatellite analysis 6 113 R For more information e hn 118 Overview of microsatellite analysis Microsatellite markers also called short tandem repeat STR markers are po
21. increases with increasing peak overlap e Spectral profile Shows the emission spectra of the dyes Raw data The emission image shows distinct fluorescence emission maxima one for each dye Figure 17 Figure 17 Example output from a spectral calibration using a matrix standard 1 Calibrated Data lt HRM E ES ES E Ex 0 400 800 1600 2000 2400 N 3200 0 Intensity vs Scan Number Intensity vs Pixel Number HEROS a g 0 2 110 120 130 140 150 160 170 180 190 200 210 220 230 240 25 DNA Fragment Analysis by Capillary Electrophoresis Q Value and Condition Number ranges Troubleshooting spectral calibration Understanding the matrix file 310 instruments only Chapter 4 Optimizing Capillary Electrophoresis Matrix standard Dye set Quality value S User provided AnyDye 0 8 default DS 30 D 0 95 DS 33 65 0 95 13 5 DS 32 F 0 95 8 5 DS 31 D 0 95 DS 02 E5 0 95 6 A poor or incorrect matrix results in too much or too little subtraction of dye spectral overlap during data analysis Each causes a recognizable electropherogram anomaly e Bleed through peaks also called pull up peaks caused by too little subtraction Elevated inter peak baseline caused by too much subtraction If the spectral calibration fails or if the quality of a passing calibration is not acceptable try one
22. plates 58 post amplification manipulations 33 primer design 29 primers preparing 57 problems caused by expired reagents 159 products storing 56 reaction volume 58 DNA Fragment Analysis by Capillary Electrophoresis reagent concentrations 59 selective amplification 31 setup 64 side reactions 60 template volume 58 time release 61 62 touchdown 61 63 troubleshooting 176 XL 63 PCR work areas 64 peak detection peak window size 94 polynomial degree 94 peak morphology troubleshooting 169 Peak Scanner Software features 92 overview 92 phylogenetic studies 110 plant genome mapping fingerprinting application 17 plant typing microsatellite application 16 Platinum Multiplex PCR Master Mix 23 24 Platinum Pfx DNA Polymerase 23 24 polymer characteristics 83 handling 83 problems caused by degraded or expired 159 types 83 polymerase enzymes See DNA polymerase enzymes polynomial degree peak detection 94 varying 95 window size value 96 pooling ratios for multiplexing 27 POP polymer See polymer population genetics studies 110 post amplification manipulations 33 preamplification troubleshooting 190 primer dimer minimizing 62 primer dimer minimizing 60 primers design guidelines 29 reconstituting 57 SNaPshot 123 tailing 34 DNA Fragment Analysis by Capillary Electrophoresis Q QF PCR relative fluorescence applications 17 143 Qualitative Fluorescence PCR See QF PCR quantification DNA template 56
23. sec Injection Time sec Resolution Increasing the injection time decreases the resolution As shown below the negative effect on resolution is more pronounced for larger fragments The decrease in resolution results from an increase in peak width as opposed to a decrease in peak separation Resolution 1 50 1 30 1 10 0 70 E 360bp range 0 50 0 30 0 10 10 20 30 40 50 60 Injection Time sec DNA Fragment Analysis by Capillary Electrophoresis 79 Optimizing injection voltage Injection voltage affects signal strength However lower voltages which produce lower currents are often preferable because injection timing is more accurate Accurate timing ensures reproducibility in sample loading Note Salt concentration can also affect signal intensity and resolution If adjusting injection time and voltage does not provide adequate signal strength you may need to concentrate the sample or desalt the sample Desalting on page 190 Attribute Effect of injection voltage Signal Peak height and peak area increase linearly with increasing injection voltage The figures below show intensity the effect of increasing the injection voltage from 53 V cm to 319 V cm on peak height and peak area respectively for two different sized fragments 1200 T 10000 T 9000 T 1000 T 2000 S soo g 7000 S H 6000 H 600 i 5000 x
24. 132 For more informatlon i cde caaled rede ri ARAR td eden e x ATR Rx dads 132 Bacterial Artificial Chromosome BAC fingerprinting 00 esses 132 Oda C IE 132 Principle of the analysis ccc cece eee e eens 133 Applications zuo e Le ner ne Lara sa ute be Reba TRU ed ERE e 133 Instrument and consumable recommendations cece eee enna 134 Experiment and primer design recommendations cece eee e ee eee 134 OTK OW terete Satie creat HARE HAEL Petes viet oid ee ee Ghar dads 135 Dataranalysiss 222 28 dos nates Pos E nO oat fich eh a ahs shee Ei We eth dia 135 For more information 0c cee eee nett tenet hee 136 High coverage expression profiling HICEP 2 00 c cc cece ccc I 136 OVERVIEW iuo aatis eret E i eee andes rcs du E tact A tn tes ta das 136 Pringiple ot the arialysiSc 4o 2 heed ot bers INC Ea tee DEA Ur 136 Application Sira an ror Ie a ei DE Le Se cae ae A DE e MIU EM 136 Recommendations cg a rg cece eect 00 R he 136 Workflow cx oo ibe ad Oe eae rs ee tet oe ETE bees 136 For more information e 9 cece cece nett mme 137 Inter simple sequence repeat ISSR PCR ence n nen ees 137 OVERVIEW 2 sco le apd ek ashley E hace Mor Ds E doa t Lor nae Gaited re pe A dci 137 Principle of the analysis 0c e eee e eens 137 Advantages cases ce oath nix ebui ee ies sande eee EBERT eS 138 Applications 2 noe rele E aie Hardee now Nietrkesgend d UL EATER RTI 138 R cormmendatlons o ors eal en ae ee enun
25. 17 What is capillary electrophoresis 18 Capillary electrophoresis CE is a process used to separate ionic fragments by size In Life Technologies CE instrumentation an electrokinetic injection is used to inject DNA fragments from solution and into each capillary During capillary electrophoresis the extension products of the PCR reaction and any other negatively charged molecules such as salt or unincorpoated primers and nucleotides enter the capillary as a result of electrokinetic injection A high voltage charge applied to the sample forces the negatively charged fragments into the capillaries The extension products are separated by size based on their total charge The electrophoretic mobility of the sample can be affected by the run conditions the buffer type concentration and pH the run temperature the amount of voltage applied and the type of polymer used Shortly before reaching the positive electrode the fluorescently labeled DNA fragments separated by size move across the path of a laser beam The laser beam causes the dyes attached to the fragments to fluoresce The dye signals are separated by a diffraction system and a CCD camera detects the fluorescence Capillary array Diffraction system Because each dye emits light at a different wavelength when excited by the laser all colors and therefore loci can be detected and distinguished in one capillary injection The fluorescence signal is
26. 34 Blue dR110 6 FAM 6 FAM 5 FAM 6 FAM 6 FAM Green dR6G HEX vic JOE vic TET Yellow dTAMRA NED NED NED NED HEX Red dROX ROX ROX ROX PET TAMRA Orange LIz LIZ t Used on 310 instruments only T Can be used for custom labeling primers DS 30 versus DS 31 VIC dye emits a stronger signal and is more stable than HEX dye Use VIC dye for weak amplicons The kits available from Life Technologies use the dye sets listed below Table 8 Dye sets and matrix standards for kits and genotyping applications Application Dye Set Raet d SNaPshot Primer Focus SNaPshot Multiplex E5 DS 02 Custom oligos D DS 30 Custom oligos Plant and Microbial AFLP Bovine and D DS 31 Canine Stockmarks Stockmarks AFLP F DS 32 Equine Stockmarks custom oligos AmpF STR9 G5 DS 33 IMPORTANT We recommend using only Life Technologies dyes Life Technologies provides spectral calibration reagents that have been optimized for our dye sets Non Life Technologies dyes or mixed isomers of dyes have variable emission spectra and also require a spectral calibration generated for the specific dyes in use to correct for the spectral overlap between the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure the dye labels do not affect PCR efficiency DNA F
27. 5 on page 35 the 5 end of the reverse primer Magnuson et al 1996 noticed a correlation between tail sequence and the amount of 3 A nucleotide addition In particular they found that adding a single G to the 5 end of the reverse PCR primer generally resulted in almost complete 3 A nucleotide addition Therefore using a tail to promote 3 A nucleotide addition can consistently yield a pattern that analysis software can identify Reverse primer tailing has advantages compared to other methods because it e Works well under diverse reaction conditions Does not require additional experimental steps Go to www lifetechnologies com for information on ordering tailed primers 34 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Figure 5 Tailed primers and 3 A nucleotide addition Labeled primer a Tailed primer ee n e 7 With tailed primers plus 7 bp Note In general the most reliable strategy is to promote 3 A nucleotide addition by modifying thermal cycling conditions and Mg concentration and if necessary by tailing the reverse primer Enzymatic treatment Ginot et al 1996 used T4 DNA polymerase to remove the 3 A overhangs from treatment pooled PCR products Although effective this method has serious limitations because it requires e A post PCR enzymatic treatment step Titrating each lot of TA DNA polymerase to determine
28. Capillary Electrophoresis Chapter 8 Fingerprinting 8 The phylograms generated from the MrBayes software is shown below It indicates with high confidence that three distinct populations of Agave shawii shawii also known as Shaw s Agave existed Figure 40 Phylogram generated using MrBayes software shows three distinct populations of Agave Individuals collected from Rosarito Arroyo Honda and Border are shown in gold grey and purple respectively Highlighted individuals correspond to the data presented in Figure 39 on page 140 Nodes in phylogram with posterior probability values above 95 are considered to be informative in Monte Carlo Markov Chain MCMC Bayesian analysis MrBayes Agave shows shavai Border LP 917 L Agave showli showll H Border J Agave showii shawii i LP 915 H Border LP 914 sor Agave shawii shawl Border 4LP 916 Agove shawii show Arroyo Honda a HLP 913 Agave shawil shawii Rosarito LP 908 Agave shawii shawii Rosarito Agave shawii showii RLP 909 Rosarito LP 910 For more For documents and publications see ISSR applications on page 200 information For ordering information see Ordering Information on page 193 DNA Fragment Analysis by Capillary Electrophoresis 141 142 DNA Fragment Analysis by Capillary Electrophoresis Overview Principle of the analysis Relative Fluorescence Quantitation RFQ BH Overview eem ASA eta ele
29. DNA Polymerase High magnesium ion concentration s AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase DNA Fragment Analysis by Capillary Electrophoresis Fluorescent labeling methods Chapter 2 Experimental Design IMPORTANT With any labeling technique use only Life Technologies dyes Life Technologies provides spectral calibration matrix standards that have been optimized for our dye sets Other dyes or mixed isomers of dyes have variable emission spectra and require a spectral calibration generated for the specific dyes to correct for the spectral overlap between the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets Table 3 Fluorescent labeling methods 5 end labeled primer incorporated during the PCR primer annealing step Fluorescent dye labeled dUTPs or dCTPs FIdNTPs incorporated during the PCR primer extension step Most commonly used in microsatellite analysis Higher precision Different fluorophores have different mobilities DNA fragments with the same 5 end primer and fluorophore have comparable electrophoretic mobility and yield sharper fragment peaks because 5 end primer labeling yields 1 1 incorporation that is one fluorophore to one DNA fragment More consistent quantitation Every peak in an electropherogram is made up of multiple DNA fragments of equal size in base pairs When using 5 end primer
30. Extra peaks troubleshooting n cece ee eee eee e eee ane 172 PER troubleshooting 2 22 4 coro eb EEUU deat d ebd Agee gee MER 176 Irregular baseline troubleshooting cece eee eee eee eens 178 Instrumentation troubleshooting 2 00 cece cee eee eee e eens 180 Sizing or Size Quality SQ troubleshooting 000 c cece eee eee ee nee e eee es 182 Viewing the size standard definition 0 00 c cece eee eee ee eee eens 182 Modifying the size standard definition ccc eee 182 Troubleshooting sanos 208 Aca tens foc ance tis Mom s at oe Fe ob hr pato Sea crie 183 GeneMapper Software troubleshooting 0 ccc c cece cece nce re 187 Preamplification gel troubleshooting cece cee eee eee eae 190 Desalting see idrico fae Cr En Asie AA LE a pare te sed Jade ee De Ada d 190 Impact of high salt concentration ccc eee eee eee 190 Eliminating salt concentration as the cause 000s 190 Desaltitig cere ee hes eek alee aber ede pe ere Ve pe eden eat I ees 190 Evaluating 310 Genetic Analyzer multicomponent matrix quality 0000055 191 Purpose of the multicomponent matrix 000000 cece eee eee eee eee 191 Factors affecting matrix quality 0 0c cece eee nena 191 When to create a new Matrix X a 9 93 e Ka 0 9 E ee ehh 191 Virtual Filter Set O 1 2 ien bt ku eed rap Ran Het Pd rete da 191 Identifying matrix problems sssssssssssssssss e 191 DNA Fragment A
31. Multiplexing Multiplexing is a PCR technique in which multiple DNA targets are amplified in the same reaction tube Multiplexing uses multiple primer pairs in each reaction and requires optimization to ensure primer pairs are compatible Life Technologies fluorescent multi color dye technology allows multiplexing Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis separates the different fluorescent dye colors into distinct spectral components Benefits Potential limitations e Simplifies PCR setup e Primer oligomer formation e Increases throughput e Loss of specificity e Decreases cost per amplification e Decreased yield of specific products Can require significant optimization 26 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Multiplexing Strategies pooling strategies Multiplexing strategies include e Pooling samples after PCR Note It is generally easier to pool the products from individually amplified fluorescently labeled primer pairs than to optimize a multiplex PCR containing multiple fluorescently labeled primer pairs Because primer efficiencies vary it may be necessary to add different amounts of each individually amplified PCR product to a pool to achieve similar peak heights Fluorescence intensity from each individual dye may also vary Amplifying multiple products in a single PCR reac
32. Standard 0 ccc ccc nnn nnn n neces 44 GeneScan 600 LIZ and GeneScan 600 LIZ v2 0 Size Standards 45 GeneScan 1200 LIZ Size Standard 0 2 0 nnna ccc ccc cence ence nen eens 47 GeneScan 350 ROX Size Standard 2 0 0 0 0c ccc ccc ccc cence nee n neces 49 GeneScan 400HD ROX Size Standard nunnana ccc cece cence tenet e ees 49 GeneScan 500 ROX Size Standard 50 GeneScan 1000 ROX Size Standard ene 51 Ordering custom primers from Life Technologies cece e cece cece eee aee 52 Testing the primers and optimizing conditions with test DNA panel 52 eG com E 52 Optimizing condition S open ee Poe SERE Chua es E 53 CHAPTER 3 Optimizing PCR 0 cece 55 Safety information cessa rede ee eel wade coavadadevendideve Sab ud hee FREE 55 Isolating purifying quantifying and storing DNA 20 cece eee ee eee eee eee 55 ISolatinq DNA ce ser tee oe ce i ae od ee a edt wee eas tices peer dec oat Sra ea 55 SH an Wd Lae BLN Aoi me en ee e de rh Lad 56 Quantifying DNA ux Re Senta Sane EXTR E een eee a vate degen 56 Storing prepared DNA before or after PCR 0000 c eee rnrn reran 56 SEINIIN E laen EIE ond ss dottor decet TEN Pt al lege pt Paiute ES 57 Reconstituting and diluting primers 0c e eee eee eee eee eee eee 57 Quantifying primers e ed Rb ee tia dE eee T beet a 57 Grel alas eyl s secre hex quee LEES kad ERE Vegas Padget duc ee ME 57
33. alleles of a single marker appear as different colored peaks at roughly the same size in the electropherogram plot The size of the different allele peaks will vary slightly due to differences in molecular weight of the dyes Figure 28 Overview of the SNaPshot kit assay Loci of interest i SS L lI 1 2 3 E 1 L A Multiplex PCR Single base extension reaction Capillary electrophoresis Electrophoretic output Relative fluorescence 3 oam Components of the system are e SNaPshot Multiplex Kit Includes SNaPshot Multiplex Ready Reaction Mix control primer mix and control template s SNaPshot Primer Focus Kit Designed to determine the approximate fragment sizes generated by various primers before SNP genotyping critical if two oligonucleotides produce overlapping signals when run simultaneously and enables the setting of tight loci windows in GeneMapper Software e GeneScan 120 LIZ Size Standard Five dye size standard that is designed for reproducible sizing of small fragment analysis data generated with the SNaPshot Multiplex Systems It accurately sizes samples ranging from 20 to 120 nucleotides nt When used with GeneMapper Software the GeneScan 120 LIZ Size Standard eliminates the need for manual genotyping Matrix Standard Set DS 02 Used for spectral calibration e GeneMapper Software Genotype analysis for data gen
34. and A T n microsatellites Nucleic Acids Res 31 974 980 Topal M D and Fresco J R 1976 Complementary base pairing and the origin of substitution mutations Nature 263 285 289 UBC Primer Set 9 2006 www michaelsmith ubc ca services NAPS Primer_Sets Primers_Oct2006 pdf Wan Q H Wu H Fujihara T and Fang S G 2004 Which genetic marker for which conservation genetics issue Electrophoresis 25 2165 2176 Wang H Z Wu Z X Lu J J et al 2009 Molecular diversity and relationships among Cymbidium goeringii cultivars based on inter simple sequence repeat ISSR markers Genetica 136 391 399 Witmer P D Doheny K F Adams M K Boehm C D et al 2003 The development of a highly informative mouse simple sequence length polymorphism SSLP marker set and construction of a mouse family free using parsimony analysis Genome Res 13 485 491 Wolfe A D Xiang Q Y and Kephart S R 1998 Assessing hybridization in natural populations of Penstemon Scrophulariaceae using hypervariable inter simple sequence repeat markers Mol Ecol 71 1107 1125 Zhao H Bughrara S S and Oliveira J A 2006 Genetic diversity in colonial bentgrass Agrostis capillaris L revealed by EcoRI Msel and PstI Msel AFLP markers Genome 49 4 328 335 DNA Fragment Analysis by Capillary Electrophoresis 205 206 DNA Fragment Analysis by Capillary Electrophoresis allele amplicon association studies backcross bin
35. as ___Pi P R et 5 0 5 x W W where P4 and P are the peak positions measured below the peak apex and W4 and W are the peak widths measured at half peak maximum An R value of 1 corresponds to fragments that can be discriminated by one nucleotide Injection time affects signal intensity and resolution Note Salt concentration can also affect signal intensity and resolution If adjusting injection time and voltage does not provide adequate signal strength you may need to concentrate the sample or desalt the sample Desalting on page 190 Attribute Effect of injection time Signal intensity Signal intensity as measured both by peak height and by peak area typically increases linearly with increasing injection time However an n fold increase in injection time does not result in an n fold increase in peak height In the examples below no improvement is seen after 10 seconds for the larger fragment The signal decreases dramatically after 40 seconds for the smaller fragment As the injection time increases the resolution decreases leading to increasing peak widths and decreasing peak heights 6000 5000 T 4000 X 3000 Peak Height FU 2000 1000 5 SS ee es ee 10 20 30 40 50 60 90000 80000 70000 a 2 Z 60000 90 amp 50000 300 e 150 340 40000 Peak Ai 30000 20000 10000 10 20 30 40 50 Injection Time
36. binset contig diploid dye set electrophoresis emission spectrum Glossary One specific sequence of a locus Different alleles of a single locus will have slightly different DNA sequences The product of a PCR reaction Typically an amplicon is a short piece of DNA Studies that interrogate a dense set of markers to identify associations between those markers and the loci of interest Association studies are more powerful than linkage mapping studies for the detection of weak susceptibility loci A genetic cross where an individual from the F1 generation is mated to an individual with the genotype of one of the parents In GeneMapper Software the expected location of an individual peak or allele defined by a base pair range and a color You typically define a bin for each possible allele associated with a marker In GeneMapper Software a set of bins that you specify in the analysis method In BAC fingerprinting a contiguous sequence of DNA created by assembling overlapping sequenced fragments of a chromosome A group of clones representing overlapping regions of the genome Having two copies of every chromosome The term dye set corresponds to The physical dye set used for labeling fragments The software selection you make that identifies the dye colors in the dye set the order of the dye peaks in the dye set and spectral analysis parameters for the dyes The act of applying an electrical fiel
37. chemicals not distributed by Life Technologies contact the chemical manufacturer Note Perform capillary electrophoresis under well controlled conditions with standard operating procedures We recommend using a dedicated instrument platform for an experiment to minimize random error due to sizing imprecision This chapter contains general information for capillary electrophoresis For application specific information on capillary electrophoresis see the application chapters later in this guide Table 10 Life Technologies Genetic Analyzers capillary electrophoresis technology Number of Capillary Polymer Data Collection Software Instrument Mgr array Sample capacity capillaries type version length 3500 8 36t and POP 7 96 well plates and 3500 Series Software v1 0 50 cm POP 49 8 tube strips or later POP 6 3500xL 24 96 and 384 well plates and 8 tube strips 3730 48 36 and POP 7 96 and 384 well plates Data Collection Software 50 cm POP 6 v3 0 or v3 1 or later 3730xl 96 3130 4 22 36 and POP 7 96 and 384 well plates Data Collection Software 50 cm POP 4 v3 0 or v3 1 or later POP 6 3130xl 16 310 1 47 cm POP 4 Up to 48 or 96 sample 310 Data Collection POP 6 tubes Software 3500 Series instruments 36 cm capillary arrays and POP 4 polymer are used for HID applications only 68 For more information on instruments see Instrument documentation o
38. definition used for analysis The GeneMapper Software includes several size standard definitions You can also create your own size standard definition files or download updated size standard definitions from our website Data from 3500 Series instruments can be analyzed with a size standard definition that specifies normalization if the data was collected with a normalization standard Size matching uses ratio matching based on relative height and distance of neighboring peaks It then derives quality values statistically by examining the similarity between the theoretical from the size standard definition and actual observed fragment patterns see the figure on the next page To complete this step successfully the analysis software must match at least three peaks The software ignores anomalous peaks that do not match the expected patterns The software constructs a best fit curve using the data points of each size standard fragment detected A comparison between the sizes calculated from the best fit curve and the matched peaks from the size standard definition using the array of numbers is performed Size matching and subsequent sizing fails if significant differences in peak patterns are found if no match can be made based on the expected patterns or if all peaks are not found Because the software uses ratio matching looks for the expected number of alleles and expected peak patterns instead of specific data points it is
39. denaturing and non denaturing applications Peaks not used for Some size standards include peaks that are not used for sizing These peaks are sizing denoted with a in the following figures These peaks can be used as an indicator of precision within a run Pre pa ring a size 1 Vortex to mix the contents of each size standard tube thoroughly then centrifuge standard briefly to collect the liquid at the bottom of the tube 2 Optimize the ratio of sample to size standard and Hi Di formamide using the values listed below as a starting point Components 3300 series 3730 series ang 310 instrument 3130 Series instruments Sample 0 5 uL per reaction 0 5 uL per reaction Size standard 0 5 uL per reaction 0 5 uL per reaction Hi Di Formamidet 9 0 uL per reaction 11 0 uL per reaction Hi Di Formamide Part no 4311320 is purchased separately from the size standard 3 Create a master mix of the size standard and formamide 4 Add samples and master mix to tubes or wells 5 Heat the reaction mix for 3 to 5 minutes at 95 C Immediately chill on ice for 2 to 3 minutes then load samples IMPORTANT After size standards are mixed with formamide run immediately Signal will decrease significantly if left at room temperature for gt 1 day or at 2 to 8 C for gt 5 days Plates can be stored at 20 C for up to 1 week DNA Fragment Analysis by Capillary Electrophoresis 43 GeneScan 120 Range 15 to 120 bp
40. ee p oen bep RES 138 Experiment and primer design considerations usann rn rnrn rreren 139 W OTK TOW paana hh en detienen Deer noe Pte del glad siete Laur bes A A 139 Data analysisS icis opp o OL Eure Ene M E Rb xA LEER 139 For more information 0c cece eect e e 141 DNA Fragment Analysis by Capillary Electrophoresis 9 Contents 10 S CHAPTER Relative Fluorescence Quantitation RFQ 143 OVERVIEW Z Ao sor A Ate et Pride Alii uc finns I LE hana A ids Rad 143 Principle of the analysiS 0 0 ccc cece AEAEE EAT R eE A 143 Leal 2 5 ener ceder tede eae pile REA RR EU een a 144 Experiment and primer design recommendations cece cece eee eee e eens 144 Recommendations zt RI baad cd dace eae hd ba eat 144 Minimizing signal intensity variation 00 cece renerne ananena 144 LOR Workflow 12e RI Rhe eit hehe tot TRR Be Gat dus PORE Vd he ee ee 145 Data analysis ce kegs Seg eee tee eo et KR bete pite dl LA ER aT 145 Precise peak detection 00 cece ere 145 Determining relative quantities 0000 cee eee eee eae 145 Determining relative number of mol cules cece eee eee eens 146 For more information seresa esius reeter ne e teen R KR ene K HR KR eee 146 Microsatellite Instability MSI and Replication Error RER 146 CHAPTER 10 Additional Applications 149 DNAimethylation emere eI rele SAR TR dE Nee due I RP eR a E Leena 149 CHAPTER 11 Troubleshooti
41. estimate the percent stutter by calculating the ratio of the combined heights of the stutter peaks with the height of the true allele peak Note the following The longer the repeat unit the less stutter product produced For microsatellite loci with the same number of repeat units the percent stutter is greater for dinucleotide microsatellite loci than it is for trinucleotide microsatellite loci and so on Walsh et al 1996 The figure below illustrates the greater stutter in dinucleotide left as compared to tetranucleotide right repeat loci Each locus is homozygous with the largest peak in each figure representing the true allele The percent stutter increases with increasing allele length that is with increasing number of repeat units However if some of the repeats are partial repeats you may not see the proportionate increase in percent stutter DNA Fragment Analysis by Capillary Electrophoresis Dinucleotide repeats Chapter 6 Microsatellite Analysis 6 Figure 23 Stutter percentages for the FGA and TH01 loci Black data points indicate loci labeled with NED dye Z 2 2 z q o S L a t tt tt ttt ttt Ft ttt 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26 29 30 3132333435 424344 45 46 47 48 49 50 5152 53 FGA Successful amplification of dinucleotide repeat markers yields allele peaks and associated stutter peaks within a maximum range of eight base pairs fr
42. freshness Buffer type and concentration Polymer type e Denaturing or non denaturing conditions e Run temperature When you create a matrix you must run each dye matrix standard separately to determine the proportional amount of fluorescence that is emitted in all four detection regions Because the emission spectra of the dyes vary with the physical environment such as the pH or polymer type and concentration create a new matrix if run conditions change If you observe any of the symptoms listed in the table on the next page create a new matrix You can apply the new matrix to old samples and reanalyze the data Matrix files made for Virtual Filter Set C are especially susceptible to minor changes in run conditions because of the emission maximum of 6 FAM dye the recommended blue displaying dye for this filter set Itis very close to the laser wavelength of 514 5 nm Thus the window for collected blue light intensity data is offset to longer wavelengths and does not contain the emission maximum of 6 FAM dye s tisalso very close to the detection region for the green displaying TET dye If you are using Virtual Filter Set C watch for evidence of matrix problems and create a new matrix as soon as problems appear A poor or incorrect matrix results in too much or too little subtraction of dye spectral overlap during data analysis Each causes a recognizable electropherogram anomaly e Bleedthrough peaks also
43. gel AFLP9 only DNA temperature settings are correct and so on Redigest DNA Desalting Impact of high salt concentration Eliminating salt concentration as the cause Desalting 190 Samples high in salt result in poor injections and low signal intensity You may be able to compensate for decreased signal intensity by Re injecting the sample Salt preferentially injects smaller fragments and inhibits injection of larger fragments so the majority of salt may have been injected in the first injection Increasing the injection time and or injection voltage If reinjecting and increasing the injection parameters does not improve signal intensity desalt and or concentrate the samples Do not increase sample concentration by evaporating the samples without performing a desalting step Doing so increases the salt concentration and prevents complete denaturation of the DNA which causes decreased signal strength To determine if salt concentration is causing low signal intensity run the size standard alone see Running controls to isolate a problem on page 156 If you see Resolution loss with the size standard troubleshoot instrument reagent issue Refer to the instrument user guide for information No resolution loss with the size standard add sample to the well containing the size standard and run again If resolution decreases desalt the sample To desalt your sample s try one of the following
44. is best to have between 50 and 200 peaks as the fingerprint after amplification 1 Restriction digestion Ligation Preselective amplification Selective amplification Capillary electrophoresis c 5 RF 9 m Data analysis DNA Fragment Analysis by Capillary Electrophoresis Data analysis Chapter 8 Fingerprinting 8 The GeneMapper Software includes an AFLP Default analysis method that you can use as a Starting point for analysis This method contains analysis parameters for pattern recognition of fragments across samples to generate a fingerprint for every sample This method can be used to analyze any type of data from fragment length polymorphism assays such as AFLP or T RFLP Features of the software useful for analysis include Ability to generate a panel the collection of markers from sample files that have been added to a project e Sizing Quality and Genotyping Quality values flag poor quality samples enabling easy identification and decrease manual review Automatic generation of final marker genotypes in a standard binary format where 1 represents the presence of a given fragment while 0 represents the absence of the corresponding fragment Up to four profiles are expected for each sample because Both the forward and reverse PCR primers may be fluorescently labeled Two restriction enzymes are used Generate panels and binsets using the AFLP Default analysis method You can then routinely an
45. labeling every DNA fragment contributes a single fluorophore to the total signal of a peak and thus the peak area is proportional to the number of DNA molecules Distinct strands By attaching different fluorophores to the forward and reverse primers it is possible to distinguish between the peaks corresponding to each strand and between residual double stranded products Lower sensitivity Because of 1 1 incorporation that is one fluorophore to one DNA fragment it yields a lower signal than FIdNTP labeled fragments Most commonly used in SNP analysis s Lower precision Fragments labeled with F dNTPs tend to produce broader peaks that often appear to be split because variable numbers of F dNTPs are incorporated during PCR in variable positions on both strands Less consistent quantitation A variable number of fluorophores are attached to each DNA fragment in a population The average number of attached fluorophores depends upon the fragment base composition and length and upon the ratio of FIdNTPs to dNTPs added to the reaction mixture Therefore it is not advisable to compare peak areas between fragments labeled with FIdNTPs for relative quantitation studies Higher sensitivity Because most fragments contain multiple fluorophores a given number of F dNTP labeled fragments will produce a higher signal when compared to the same number of 5 end labeled fragments The increased signal strength allows y
46. largest sample fragments of interest The set of size standard peaks that GeneMapper Software uses to generate the sizing curve can vary with the size calling method In general be sure to include the two size standard peaks immediately smaller than the smallest fragment and the two size standard peaks immediately larger than the largest sample fragment of interest or modify the size standard definition to eliminate the peaks that are not present Note For faster run times you can also increase the run voltage However a higher run voltage can decrease the resolution Optimizing Run voltage can affect migration rates and resolution because it affects the speed at run voltage which samples migrate through the capillary If they migrate too quickly the samples do not optimally separate Attribute Effect of run voltage Migration Higher run voltages yield faster run times but can affect the resolution rates 140 j s too a a 13 5 13 8 12 5 12 8 11 5 11 8 16 5 16 8 9 5 9 8 8 5 8 8 7 5 7 8 6 5 6 8 55 5 8 L 1 1 L l 1 l 240 260 280 308 320 AG 368 o 204 bp Fragment e 5117 bp Fragnent Minutes o TTTTTTTTTTT71717771 7T o iS See ee oe ee ee H ee ee ee ee Viem Resolution In general resolution is better at lower run voltages 24 o 22r o o 280r o o 190 204 bp 508 554 bp Resdutian bh o o T T T N T esr e6r 240 260 280 300 320 AG 368 Yi
47. libraries In Ferraris J and Palumbi S eds Molecular Zoology Advances Strategies and Protocols Wiley Liss NY 459 468 Goel S Chen Z Akiyama Y Connor J A et al 2006 Comparative physical mapping of the apospory specific genomic region in two apomictic grasses Pennisetum squamulatum and Cenchru sciliaris Genetics 173 1 389 400 Gupta S K Souframanien J and Gopalakrishna T 2008 Construction of a genetic linkage map of black gram Vigna mungo L Hepper based on molecular markers and comparative studies Genome 51 628 637 DNA Fragment Analysis by Capillary Electrophoresis 203 204 Hauge X Y and Litt M 1993 A study of the origin of shadow bands seen when typing dinucleotide repeat polymorphisms by the PCR Hum Mol Genet 2 411 415 Holleley C E and Geerts P G 2009 Multiplex Manager 1 0 a cross platform computer program that plans and optimizes multiplex PCR BioTechniques 46 511 517 Huelsenbeck J P and Ronquist F 2001 MRBAYES Bayesian inference of phylogeny Bioinformatics 17 754 755 Inazuka M Tahira T and Hayashi K 1996 One tube post PCR fluorescent labeling of DNA fragments Genome Res 6 551 557 Inazuka M Wenz H M Sakabe M Tahira T and Hayashi K 1997 A streamlined mutation detection system multicolor post PCR fluorescence labeling and single strand conformational polymorphism analysis by capillary electrophoresis Genome Res 7 1094 1103
48. mO and c are calculated by a least squares fit to minimize X 2i Li e mi m0 LO the left side quantity How the Global Southern method works All points in the standard are weighted equally and the curve is not constrained to pass through any specific point The software can analyze a large range of fragment sizes with this method For best results use a size standard that has at least two peaks smaller than the smallest fragment of interest and at least two peaks larger than the largest fragment of interest DNA Fragment Analysis by Capillary Electrophoresis 103 Evaluating data quality Examining PQVs Criteria for a good electropherogram 104 Note For detailed information on quality value determination see the GeneMapper Software Reference and Troubleshooting Guide v4 1 Pub no 4403673 The GeneMapper Software displays Process Quality Values PQVs in the Samples or Genotypes tab of the Project window Symbol Definition Bersnt Range K Pass The sample or genotype passed the PQV test 0 75 to 1 0 A Check A possible problem exists for the sample or 0 25 to 0 75 genotype 9 Low Quality Fail There is a strong possibility that a 0 0 to 0 25 problem exists for the sample or genotype Review the SQ Sizing Quality and GQ Genotype Quality results for each sample Many of the PQVs can affect the GQ result Note If the SQ PQV is the sample is not sized or genoty
49. of non specific products background Sudden loss of signal in all samples Multiple dye colors are detected as one dye color Instrument laser power or current problem 5 dye samples were run with a 4 dye matrix Check laser power and current in the EPT window see Examine the sample info raw data and EPT trace on page 154 Repeat run with correct dye set DNA Fragment Analysis by Capillary Electrophoresis 171 Extra peaks troubleshooting Symptom Possible cause Action Extra peaks Pull up or cross talk due to saturated data in a dye color for example a high intensity blue peak can create pull up peaks in other colors See Signal intensity is too high or saturated on page 166 Decrease sample concentration during PCR or when preparing samples for electrophoresis Degraded PCR products Repeat PCR Stutter peaks See Identifying stutter products in microsatellite analysis on page 113 Incomplete restriction or ligation AFLP applications only Extract the DNA again and repeat the restriction ligation Sample is not denatured Make sure the samples are heated at 95 C for 3 to 5 minutes then immediately placed on ice for 2to 3 minutes before loading Hairpin secondary structures are present in PCR primers Non specific primer peaks See PCR troubleshooting on page 176 Primer dimer peaks See Primer design guidel
50. optimal enzyme concentrations and treatment times IMPORTANT Excess T4 treatment can cause PCR product degradation Insufficient treatment will not remove the 3 overhangs and can make some alleles more difficult to genotype Start optimization experiments with 0 5 to 1 unit of TA DNA polymerase in 10 uL of pooled PCR product Incubate at 37 C for 30 minutes DNA Fragment Analysis by Capillary Electrophoresis 35 Dyes Dyes and chemical forms IMPORTANT We recommend using only Life Technologies dyes Life Technologies provides spectral calibration reagents that have been optimized for our dye sets Other dyes or mixed isomers of dyes have variable emission spectra and also require a spectral calibration generated for the specific dyes in use to correct for the spectral overlap between the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets Life Technologies dyes are available in multiple chemical forms Some forms are supplied coupled to primers and others you can use to label custom primers or fragments Each form has distinct advantages and disadvantages depending upon the intended application and your laboratory setup You can analyze phosphoramidite labeled fragments with NHS ester labeled fragments but you should not combine F dNTP labeled fragments with any other labeling method Table 5 Dye chemical forms Chemical form Purpose Available
51. peak height DNA Fragment Analysis by Capillary Electrophoresis LOH workflow Data analysis Precise peak detection Determining relative quantities Chapter 9 Relative Fluorescence Quantitation RFQ 9 For more information see Optimizing signal intensity on page 77 Trregular signal intensity troubleshooting on page 164 Relative fluorescence quantitation applications on page 200 IMPORTANT Preferential amplification can decrease the accuracy of relative quantitation measurements For more information see Preferential amplification on page 31 1 Design the primers and select the primer dye set 2 PCR Runtwo DNA samples from each individual for example One from normal tissue N One from tumor tissue T Note Some normal tissue contaminating the tumor tissue sample is typical e Run3to4 independent injections for each sample N and T to obtain sufficiently accurate quantitative estimates for subsequent data analysis s Run control DNA Amplify at least one control DNA sample for every round of PCR amplification Run at least one injection of amplified control DNA for every set of microsatellite markers used Run at least one injection of amplified control DNA whenever you change the capillary or electrophoresis conditions 3 Capillary electrophoresis 4 Data analysis Optimize peak detection parameters to ensure precise peak detection For more information see Data Ana
52. primers 57 Quantitative Multiplex PCR of Short Fluorescent Frag ments See QMPSF R raw data examine 154 example 155 reagent troubleshooting 158 159 Relative Fluorescence Quantitation See RFQ Replication error 146 RER 146 resolution definition of 79 formula 79 troubleshooting 169 RFQ applications 17 144 data analysis 145 determining relative number of molecules 146 determining relative quantities 145 experiment and primer design recommendations 144 LOH workflow 145 minimizing signal intensity variation 144 overview 143 peak height versus area 143 RFU ranges for instruments 158 rTth DNA polymerase description 24 thermal cycler parameters 63 XL 24 run modules 46 S Safety Data Sheets SDSs obtaining 201 safety information 55 salt concentration desalting 190 impact on electrophoresis 82 sample concentration problems caused by high 158 215 contamination problems caused by 158 sample salt concentration See salt concentration secondary structure primer 30 template 31 selective amplification 31 signal intensity minimizing variation 144 optimizing 77 range for each instrument 77 158 relative order of dyes 38 troubleshooting 164 Single Nucleotide Polymorphism See SNP singleplexing 26 Size Match Editor displaying 153 size methods GeneMapper Software 100 size standards function 42 GS1000 ROX 51 GS120 LIZ 44 GS1200LIZ 47 GS350 ROX 49 GS400HD ROX 49 GS500 LIZ 44 GS500 ROX 50 GS600 L
53. primers that flank the region of interest PCR fragments of different sizes are generated based on the length of the dinucleotide repeat Figure 19 Different repeats lead to PCR fragments of different length arrows indicate forward and reverse primers Size in bps 150 GAJCA CA s 152 X CA CA CA CA q 154 CACA cA caca Several features of microsatellites and their corresponding set of alleles make them ideal for use in genetic studies e They are present in large numbers e They are relatively evenly spaced throughout the genome and often physically situated near or within genes e They show a varying but relatively high mutation rate relative to non microsatellite loci Mitochondrial DNA evolves 5 to 10 times faster than single copy nuclear DNA Microsatellites evolve 100 to 1000 times faster than single copy nuclear DNA The mutation rate of microsatellite loci is 10 to 10 6 events per locus per generation Wan et al 2004 The rate is believed to be different depending on the number of nucleotides in the repeated unit Eckert and Hile 2009 DNA Fragment Analysis by Capillary Electrophoresis Chapter 6 Microsatellite Analysis S Their alleles are inherited in a Mendelian manner and are stable over multiple generations Their alleles can be unique to specific populations Detailed data on allelic variation number of repeats and allelic frequencies are
54. purification Digestion Adaptor ligation Selective PCR Post PCR preparation Capillary electrophoresis Do SO he Oe SO SESS COUP nS Data analysis DNA Fragment Analysis by Capillary Electrophoresis Chapter 8 Fingerprinting 8 Figure 35 HiCEP workflow MAAAAA AAAAAA Ba AAMAS avidin mn BABAR avidin i B B TTTTTT biotin ITTTTT biotin ESI ITTTTT biolin ril TTTTTTebiotin EI x Double stranded cDNA is synthesized The double stranded cDNA is digested 1 Msp or Mse adaptors are ligated to A second round of Msel or Msp Msp lor Msel adaptors are ligated to using purified paty A RNA and a with a amp bp recagnition site restriction each fragment restriction digestion is performed each fragment biotinylated oligoldT primer endonuclease Msp lor Msel 2 Biotinylated fragments are purified using avidineconjugated magnetic beads M 6 Selective PCR 7 Post PCR preparation 8 Capillary electrophoresis T zm S CE mmm T S E bs ol l detectable undetectable U T Seloctive PCR is performed with a set of 256 Dilutions of the completed HiCEP reactions Data are analyzed using GeneMapper primers which cover the Msel and Mspi are combined with the Applied Biosyste Software version 4 1 adaptor sequences and all combinations of GeneScan 600 LIZ Size Standard vers the two nucleotides NN adjacent to them 2 0 and Hi Di Formamide and denatured 3500xL Genetic Analyzer with
55. reactions You will often need to optimize reaction conditions and occasionally you will need to redesign the primers Troubleshooting multiplex PCR Consider amplifying separately any primer pair that fails to amplify after its concentration is increased To eliminate interfering background peaks try e Swapping primer pairs between different multiplex reactions Removing primer pairs from the multiplex reaction Primer design guidelines Primer design Optimum length 17 to 25 nucleotides criteria e Optimum Tm 55 to 65 C Using primers with similar T4 values makes it possible to find thermal cycling parameters that are optimal for both primers in a primer pair The Tn of a reaction is influenced by base composition concentrations of Mg and K ions in the mixture and cosolvents Based on the T calculate the annealing temperature Ta CC T4 5 The 244 Rule T4 A T X 2 G C X4 Avoid Primer dimers Hairpins Secondary structures Secondary binding sites Primer design Life Technologies offers OligoPerfect Designer available at software www lifetechnologies com DNA Fragment Analysis by Capillary Electrophoresis 29 Factors affecting Tm and primer annealing 30 Primer annealing is influenced by Primer template base composition e Primer template base order Primer or template secondary structure Effects of base composition G C bonds contribute more to the stability increased melt
56. than expected Amplification cycle setting is too low Low MgCl concentration Add 3 to 5 cycles Increase the MgCl concentration Low affinity of the primer to the template Decrease the annealing temperature 2 to 3 C at a time Background signal may increase Low sample concentration Increase sample concentration Inhibitors in template Purify template see Purifying DNA on page 56 Thermal cycler malfunction Troubleshoot the thermal cycler problem Refer to the thermal cycler user guide for information PCR reagents are contaminated or expired Use fresh PCR reagents Degraded primers Store unused primers at 15 to 25 C Do not expose fluorescent dye labeled primers to light for long periods of time PCR inhibition e Sample contains hemoglobin heparin polyphenol plant polysaccharides Extraction introduced inhibitors chloroform phenol EDTA detergents SDS xylol ethanol bromophenol blue Dilute the sample before amplification to reduce the amount of PCR inhibitors Contamination with exogenous DNA Carryover Use appropriate techniques to avoid introducing foreign DNA during laboratory handling For more information see Avoiding contamination on page 64 176 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible Cause Action Poor amplification non spec
57. to light for long periods of time Size standard signal and sample signal are not balanced o 3200 300 3 0 3508 3900 700 Size standard concentration is too high e Although the data is still sized properly decrease size standard concentration to balance peaks in future runs see Size standard peak intensity on page 42 DNA Fragment Analysis by Capillary Electrophoresis 167 Migration troubleshooting Symptom Possible Cause Action Size standard peaks are not migrating as expected during a normal run time Poor quality sample e Degraded or frozen polymer e Water used to dilute buffe r e Poor quality formamide e Fluctuations in ambient temperature and or humidity Incorrect oven temperature e Old array or capillary Contaminants e Low ionic buffer strength Prepare fresh buffer not applicable to 3500 Series instruments Incorrect capillary length Length to Detector or run module was selected Specify correct capillary length or run module Variation in ambient temperature causes faster or slower migration rates Ensure ambient temperature is stable Sizing precision is low Analyzing small fragments 50 bp e Sizing differences between various types of polymer are more apparent for sequences 50 bp e Fragments 50 bp run on 3730 3730xl Series instruments with POP 77 po
58. to nonspecific amplification particularly if coupled to poor quality g DNA extraction methods and suboptimal PCR amplification conditions Principle of the ISSR PCR uses a single fluorescently labeled primer to target the region between analysis identical microsatellites Figure 36 An ISSR PCR primer comprises three parts e A fluorescent tag Eight dinucleotide repeat units or 6 trinucleotide repeat units One or more anchor nucleotides designed with a dual purpose to target the end of a microsatellite region and to prevent primer dimerization More than 100 primers have been developed for use in ISSR techniques UBC Primer Set 9 2006 catalog DNA Fragment Analysis by Capillary Electrophoresis 137 Advantages Applications Recommendations 138 Figure 36 Example inter simple sequence repeat ISSR region dye e FAM 39767 GT GT GT GTGT GTA a GT repeat anchor 5 GTGTGTGTGTGTGTGTGTGT ACACACACACACACACACAC 3 3 CACACACACACACACACACA TGTGTGTGTGTGTGTGTGTG 5 Because ISSRs are dominant markers the amplified regions in an ISSR PCR are scored as diallelic Between individuals within a population changes in the amplified products can arise through structural changes to the region insertions or deletions or the loss of primer binding sites Faster and requires a lower startup investment than other genotyping methodologies such as AFLP and RFLP Several studies have compared AFLP and ISSR res
59. tty vue ees ES 143 m Experiment and primer design recommendations 0000 eee 144 B LOH workflow eter ger odes ede de de e VAS 145 E Data analysisis 3 tseh ere Petey Peas cnt ned E RRR NR PUR e e T 145 B For more information cies esee e E KR hy ober dba alot a died RR NE ES 146 Microsatellite Instability MSI and Replication Error RER 146 Relative fluorescence quantitation RFQ is a technique used in a variety of fragment analysis applications to compare peak heights across samples Relative fluorescence applications compare peak height or area between two samples Common techniques include Qualitative Fluorescence QF PCR Quantitative Multiplex PCR of Short Fluorescent Fragments OMPSF Multiplex Ligation dependent Probe Amplification MLPA The data for an RFQ experiment can be obtained with microsatellite or AFLP analysis Peak height or peak area can be used to compare differences in the same marker across multiple samples However you may see a difference in results depending on whether peak height or peak area is used IMPORTANT Variations in signal intensity adversely affects results in RFQ experiments For information on minimizing variation see Optimizing signal intensity on page 77 As an example microsatellite RFQ experiment the figure below shows an electropherogram of a microsatellite marker in DNA from a healthy and tumor sample The reduced peak height in the tumor sample in
60. under denaturing conditions LIZ Size Standard GeneScan 120 LIZ denatured fragment lengths nt 9 fragments 15 35 80 20 50 110 25 62 120 This single stranded size standard was designed to provide accurate sizing of short DNA fragments Therefore it is particularly useful for SNP analysis All fragments have been optimized under a wide variety of run conditions Figure 8 GeneScan 120 Size Standard run under denaturing conditions GeneScan Range 35 to 500 bp under denaturing conditions 900 LIZ Size This size standard is recommended for analysis of tri and tetranucleotide Standard microsatellite loci which can often exceed 400 bp in length GeneScan 500 LIZ denatured fragment lengths nt 16 fragments 35 139 250t 400 50 150 300 450 75 160 340 490 100 200 350 500 Do not use this fragment for sizing See Peaks not used for sizing on page 43 for information Only one strand of the double stranded DNA fragments in this size standard is labeled The unlabeled strand does not interfere with peak detection of the labeled strand when run under denaturing conditions 44 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Figure 9 GeneScan 500 LIZ Size Standard run under denaturing conditions CS GeneScan 600 Note The GeneScan 600 LIZ and GeneScan 600
61. 0 314 600 880 60 320 614 900 80 340 620 920 100 360 640 940 114 380 660 960 120 400 680 980 140 414 700 1000 160 420 714 1020 180 440 720 1040 200 460 740 1060 214 480 760 1080 220 500 780 1100 240 514 800 1120 250 520 820 1160 260 540 840 1200 The high fragment density 68 fragments yields greater sizing precision and landmark fragments allow easy peak pattern identification during data analysis This size standard is ideal for BAC fingerprinting T RFLP VNTR STR and many other DNA fragment analysis applications Figure 11 GeneScan 1200 LIZ Size Standard run under denaturing conditions DNA Fragment Analysis by Capillary Electrophoresis 47 48 Downloading 3130 instrument run modules from our website Updated run modules for the 3130 Series instrument and the GeneScan 1200 LIZ Size Standard are available on our website Before using the downloaded run modules adjust as described below Further optimization may be necessary 36 cm array with POP 49 polymer 50 cm array with POP 49 polymer Decrease run voltage to 8000 volts Decrease run voltage to 12 000 volts ncrease run time to 6000 seconds ncrease run time to 6000 seconds Downloading 3730 instrument run modules from our website Updated run modules for the 3730 Series instrument and the GeneScan 1200 LIZ Size Standard are
62. 0 Series and 3130 Series instruments e Sample volume must be 212 uL for 310 instruments Autosampler is misaligned e 3500 Series 3730 Series and 3130 Series instruments Fill wells with 0 5 uL size standard and 9 5 uL sample then re inject If the signal is still missing contact Life Technologies e 310 instruments Recalibrate autosampler DNA Fragment Analysis by Capillary Electrophoresis 165 Symptom Possible cause Action Signal intensity is too high or Raw data pull down peaks saturated Sample concentration too high Decrease sample concentration Decrease injection time Ski slope peak pattern of sample peaks but size standard peak heights do not decrease Sample concentration too high Optimize ratio of DNA template and primer in amplification step or insufficient primer is present 166 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible cause Action Decreased signal Degraded or improperly stored Hi Di Formamide Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 Expired or incorrectly stored reagents Use fresh reagents Degraded primers Store unused primers at 15 to 25 C Do not expose fluorescent dye labeled primers
63. 06 and Johnson et al 2005 Phylogeny studies Goel et al 2006 Breeding Zhao et al ibid e Backcross studies Johnson et al and Goel et al ibid Mapping of cloned fragments in bacterial and yeast artificial chromosomes BACs and YACs Serra et al 2006 Naimuddin et al 2004 Identifying new species or subspecies Johnson et al ibid and Savelkoul et al 1999 The AFLP kits available from Life Technologies are optimized for plants and microbes However they are an excellent starting point for custom AFLP experiments on other organisms such as fish Contact your Life Technologies field applications specialist for more information on using Applied Biosystems AFLP kits to conduct experiments in organisms other than plants or microbes This is a Life Technologies supported protocol Thermal cycler Veriti standard mode only GeneAmp 9700 2720 Genetic analyzer 3500 Series 3730 Series 3130 Series or 310 instruments Polymer see Run modules on page 69 for the polymer and capillary array length combinations supported on each instrument e Capillary array 3500 Series instruments 50 cm 3730 Series 3130 Series and 310 instruments 36 cm s GeneScan 500 ROX Size Standard included in kits e DS 32 Matrix Standard Dye Set F IMPORTANT Throughout a set of experiments use all the same equipment run parameters polymers dyes and so on Consistent conditions are required to
64. 1 4000 e 1 9 amp 3000 209 9 150 pes orf 4 0 T 4 25 50 75 10 125 15 25 5 0 75 10 125 15 Electric Field kV Electric Field kV Resolution Injection voltage has little effect on peak resolution 1 60 1 50 e 1 40 e 3 e i 160bp range g 110 E 360bp range 100 0 90 0 80 25 5 0 75 10 125 15 Electric Field kV Optimizing electrophoresis conditions 80 Optimizing electrophoresis conditions run time run voltage run temperature can greatly improve data quality run to run precision and or throughput Optimize settings appropriate for Range of fragment lengths Required degree of resolution Type of genetic analysis you will be performing for example denaturing or non denaturing conditions The settings in the run modules provided are set to ensure the following Detection of all fragments in the typical size range for the application e Acceptable run times e Acceptable resolution DNA Fragment Analysis by Capillary Electrophoresis Chapter 4 Optimizing Capillary Electrophoresis Optimizing Perform trial runs to determine the minimum acceptable run time for a given run run time voltage To ensure that you collect sufficient data to perform analysis set the electrophoresis run time approximately 10 higher than the migration time of the largest fragment of interest The largest fragment of interest is often a size standard peak that is needed for sizing the
65. 384 well small sample volume System 9700 blocks Auto Lid Dual 2 aluminum 384 well plate No 5 to 20 uL high throughput 384 Well 0 02 mL 384 well small sample volume with GeneAmp PCR blocks robotic capability System 9700 2720 Thermal 0 2 mL aluminum 96 well 0 2 mL format No Ideal for basic PCR using Cycler single block 0 2 mL reaction tubes or 96 well reaction plates 58 DNA Fragment Analysis by Capillary Electrophoresis Chapter 3 Optimizing PCR Reagent concentrations dNTP concentration Magnesium ion Template concentration The following factors can affect overall yield of specific DNA target sequences dNTP concentration Magnesium ion concentration Primer concentration Template concentration Enzyme concentration In the standard GeneAmp PCR protocol the concentration of each deoxynucleoside triphosphate dNTP is 200 uM In most cases lower dNTP concentrations do not significantly affect the yield of PCR amplification product and will increase the fidelity of the PCR amplification product However for efficient base incorporation keep the four dNTP concentrations balanced and above the estimated Kn of each dNTP 10 to 15 uM Some applications might require higher dNTP concentration especially when dNTP analogues are used However excess dNTPs decrease enzyme fidelity DNA polymerases require free magnesium ion in solution for activity For most PCR amplifications you ca
66. 5 dye set so you would also need the DS 33 matrix standard for spectral calibration see Table 7 on page 41 for the matrix standard that corresponds to each dye set Note that different dyes can be used for similar fragment lengths and the same dye can be used for fragments of different lengths Locus iad Dye Marker 1 90 104 6 FAM Marker 2 112 146 vic Marker 3 119 177 PET Marker 4 117 202 NED Marker 5 156 190 6 FAM Marker 6 221 253 6 FAM Marker 7 234 282 NED Marker 8 260 342 vic Marker 9 311 327 6 FAM Marker10 340 380 NED Capillary electrophoresis array view of example 10 marker panel DNA Fragment Analysis by Capillary Electrophoresis Dye sets Dye sets and matrix standards Creating a custom dye set Chapter 2 Experimental Design A dye set corresponds to the group of dyes you select for labeling described in the previous section You use the matrix standard that corresponds to the dye set shown below to perform a spectral calibration This calibration prepares the instrument for detection of the dyes with which your primers are labeled For information on spectral calibration see Understanding spectral calibration on page 84 Table 7 Dye set and matrix standard components Dye Set ROX LIZ8 and TAMRA dyes are reserved for the size standard Dye Set E5 D D F G5 ct Saet j D9 02 ps 30t8 DS 318 DS 32 DS 338 DS
67. CTAGA C dTAMRA Yellow Xhol C TCGAG T dROX Red Haelll GG CC None Applications With BAC fingerprinting you can create whole genome physical maps that are important resources for Genome sequencing Positional cloning Comparative genomics DNA Fragment Analysis by Capillary Electrophoresis 133 Instrument and consumable recommendations Experiment and primer design recommendations 134 This is a Life Technologies demonstrated protocol IMPORTANT BAC fingerprinting is based upon pattern recognition therefore data analysis is focused on relative size and distribution We recommend using a dedicated instrument platform to minimize low random error caused by sizing imprecision Thermal cycler Veriti GeneAmp 9700 2720 Genetic analyzer 3500 Series 3730 Series 3130 Series and 310 instruments Polymer see Run modules on page 69 for the polymer and capillary array length combinations supported on each instrument Capillary array 3500 Series instruments 50 cm 3730 Series 3130 Series and 310 instruments 36 cm GeneScan 120 LIZ Size Standard DS 33 Matrix Standard Dye Set G5 IMPORTANT Throughout a set of experiments use all the same equipment run parameters polymers dyes and so on Consistent conditions are required to avoid mobility shifts that interfere with accurate interpretation of data Protocols may differ based on the kind of restriction endonucleases and th
68. DNA Fragment Analysis by Capillary Electrophoresis Contents Using controL DNA ele eta Wink ees he ee Wide ae ae Pes PAE 57 Purpose of control DNA rentre Hae REMERPRID TALE o py E a ded 57 Guidelines for use enc ex M e eb Sade ERR de 57 CEPH 1347 02 Control DNA 0 20 cece cece I re 57 Reaction volumes and plate types n 0 cece see 58 Reagon volumes coe Z aa Ta text x m e tese eie ha ioe 58 Using small amounts of template 000 eee eee eee eee 58 Plate types x oss ent Lupe eRRERD IO GREER EC a ee ue cadet ore dat 58 Reagent concentrations 2 ccc cc seen 59 dNT Pconcentration ss s cui eos e pot Ce eatin aE ENSE E eee eres Bowes 59 Magne Umie aeae AEE ate see A ute D dm 59 Template concentration eraan aa aa a ehh 59 Enzyme concentration rs reren ee dae AR ERE A EIER EA RE N N 60 Preventing competing side reactions hot start PCR cece eee e eee eee 60 WHeMMO USC oc fii EE tearet baad deseo ended Sale A peni Pub deed HIEMIS 60 Limitations and alternatives lt c 2 7 T rriat n onae 2 Ta 3 0 me 60 Thermal cycling parameters Veriti Thermal Cyclers 000 ccc cece cence ence nee 60 Arnplifag Gold eia ber beue e ditti 4a H PEE A ee aks 60 General PCR eeen nace une sedere E wee Sea E ace ER E SR del Yee eae 61 Timesrelease PCR bs nd tee ed eee eee ad ae ee ENS 61 Touchdowr P6 ac ste cousin avit eiiis qr E sos ee NECS Caan eee ay 61 Thermal cycling parameters 9700 Thermal Cyclers
69. HiCEP Overview Principle of the analysis Applications Recommendations Workflow 136 The high coverage expression profiling HiCEP method of fragment analysis was developed to address the shortcomings in gene expression profiling and to provide a sensitive method for detecting a large proportion of transcripts in both known and unknown genes with a low false positive rate As an AFLP based gene expression profiling method the HiCEP method does not require sequence information and has a reduced rate of false positives with a high degree of detection of both coding and noncoding transcripts After HiCEP analysis fragments of interest can be purified and cloned from agarose gels and sequenced to identify the transcripts If whole genome sequence information for the organism under study is known the fragments of interest can be identified by bioinformatic prediction using the sequence information available from public databases and the restriction enzyme recognition sites used in the HiCEP workflow HiCEP is an AFLP based method The analysis involves digesting genomic DNA to produce a population of restriction fragments Priming sites are then ligated onto the ends of the restriction fragments so that they can be amplified by PCR Goel et al 2006 Fingerprinting This is a Customer demonstrated protocol For information refer to HiCEP applications on page 200 Synthesis Digestion Adaptor ligation and
70. IZ 45 GS600 LIZ v2 0 45 modifying definition 153 normalization 45 preparation 43 storage 43 troubleshooting 182 size calling guidelines 90 sizing Cubic Spline method 101 curve 100 Global Southern method 103 how the GeneMapper Software performs 98 Least Squares method 100 Local Southern method 102 methods 100 troubleshooting 153 182 sizing quality checking 153 troubleshooting 182 software analysis 89 spatial calibration overview 84 spectral calibration 216 matrix standards 41 overview 84 split peaks incomplete 3 A addition 33 SQ See sizing quality standards See also size standards 42 install standard for troubleshooting 157 matrix for spectral calibration 86 stutter identifying 113 troubleshooting 113 SuperScript III Reverse Transcriptase 23 24 support obtaining 201 T tailing primer 34 technical support 201 template DNA 56 59 small volume 58 terminal restriction fragment length polymorphism See T RFLP thermal cycler parameters general PCR 61 62 hot start 60 62 linkage mapping 62 optimizing 63 time release PCR 61 62 touchdown PCR 61 63 XL PCR 63 thermal cyclers applications 58 safety information 55 specifications 58 time release PCR enzyme 23 thermal cycler parameters 61 62 Tm calculator 29 factors affecting 30 primer 29 touchdown PCR thermal cycler parameters 61 63 training information on 201 T RFLP applications 131 data analysis 132 experiment and primer desig
71. LIZ v2 0 Size Standards contain LIZ and the same peaks The GeneScan 600 LIZ v2 0 Size Standard can be used for GeneScan 600 normalization on 3500 Series instruments LIZ v2 0 Size Range 20 to 600 bp under denaturing conditions Standards GeneScan 600 LIZ denatured fragment lengths nt 36 fragments 20 120 220 314 414 514 40 140 240 320 420 520 60 160 250 340 440 540 80 180 260 360 460 560 100 200 280 380 480 580 114 214 300 400 500 600 DNA Fragment Analysis by Capillary Electrophoresis 45 46 Figure 10 GeneScan 600 LIZ Size Standard fragments run under denaturing conditions Optimizing the 3130 Series instrument run module The run modules provided with these instruments may need to be optimized for use with the GeneScan 600 LIZ Size Standard Add 100 seconds to the run time if needed Optimizing the 310 instrument run module The run modules provided with these instruments have not been optimized for use with the GeneScan 600 LIZ Size Standard Add 200 seconds to the run time before using this size standard DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design GeneScan 1200 Range 20 to 1200 bp under denaturing conditions LIZ Size Standard GeneScan 1200 LIZ Size Standard denatured fragment lengths nt 68 fragments 20 280 560 850 30 300 580 860 4
72. Note Sample overloading can clog capillaries e Optimize the ratio of sample to size standard and Hi Di formamide using the values listed below as a starting point Components 3500 Series 3730 Series and 3130 Series instruments 310 instrument Sample Size standard 0 5 UL per reaction 0 5 UL per reaction 0 5 UL per reaction 0 5 UL per reaction Hi Di Formamidet 9 0 uL per reaction 11 0 uL per reaction t Hi Di Formamide Part no 4311320 is purchased separately from the size standard e Ifyou anticipate an extremely high sample concentration run dilutions of the sample If the signal is too strong you can further dilute the sample or you can decrease the sample injection time and or injection voltage e Ifthe signal is too weak first try increasing the signal by increasing the sample injection time or voltage To optimize the signal intensity for a given sample inject the same sample multiple times using a range of injection parameters If the signal intensity is still too weak or the resolution is poor concentrate the sample DNA Fragment Analysis by Capillary Electrophoresis Optimizing signal intensity Optimal detection ranges Balancing size standard and sample peak intensities Chapter 4 Optimizing Capillary Electrophoresis If the signal intensity is too low after concentration see Desalting on page 190 Different dyes emit different fluorescenc
73. PCR preventing mispriming and enhancing PCR specificity and yield AmpliTaq DNA Polymerase For general use in PCR AmpliTaq9 DNA Polymerase is a recombinant form of Taq DNA polymerase obtained by expressing a modified Taq DNA polymerase gene in an E coli host Similar to native Taq DNA polymerase the enzyme lacks endonuclease and 3 5 exonuclease proofreading activities but has a 5 3 exonuclease activity AmpliTaq DNA Polymerase LD Low concentrations of E coli DNA contamination thus is better suited for amplifying DNA of bacterial origin AmpliTaq DNA Polymerase LD Low DNA is the same enzyme as AmpliTaq DNA Polymerase however the LD formulation has undergone a further purification process The purification step insures that false positive PCR products will be effectively minimized when amplifying bacterial sequences AmpliTaq DNA Polymerase LD is especially useful for low copy number amplifications 22 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design DNA polymerases Description AmpiTaq Gold DNA Use in most applications because it yields PCR fragments of high specificity Poymerase AmpliTaq Gold DNA Polymerase is a chemically modified form of AmpliTaq DNA Polymerase It provides the benefits of hot start PCR that is higher specific product yield increased sensitivity and success with multiplex PCR without the extra steps and modification
74. POP 7 at 95 C for 5 min polymer and a 50 cm array The use of a bp recognition site restriction enzyme generates small fragments which is important for improving subsequent PCR amplification efficiencies f Mspl is used far the first digestion Msel is used for the second digestion and vice versa HT n For more For documents and publications see HiCEP applications on page 200 information For ordering information see Ordering Information on page 193 Inter simple sequence repeat ISSR PCR Overview Inter simple sequence repeat ISSR PCR is a fast and inexpensive genotyping technique with a wide range of uses including the characterization of genetic relatedness among populations ISSR PCR is a genotyping technique based on variation found in the regions between microsatellites In addition to the use of long fragments for accurate analysis this technique provides additional benefits over agarose gels The increased sensitivity of Life Technologies genetic analyzers over traditional analysis methods routinely allows the detection of an order of magnitude more peaks and this increased resolution results in better discrimination between individuals being compared in the populations However the primers that are designed to anneal to the di or trinucleotide repeats can lack specificity in PCR and are a major contributor to a lack of reproducibility Also the lack of complexity of the ISSR primers can lead
75. S 1 887158E 09 R 2 1 000 Size Calling Curve 3rd Order Least Squares 1000 Cubic Spline Interpolation method DNA Fragment Analysis by Capillary Electrophoresis T T T T T T T k T T T T T T T T 1500 2000 2500 3000 3500 4000 4500 1000 1500 2000 2500 3000 3500 4000 4500 The Cubic Spline method forces the sizing curve through all the known points of the selected size standard Although this enforcement produces exact results for the values of the standards themselves it does not compensate for standard fragments that may run anomalously Cubic Spline interpolation sizing curve Best Fit 2nd Order Curve A0 9 771623E 01 Al 1 220838E 01 A2 4 856885E 06 R 2 1 000 Size Calling Curve Cubic Spline Interpolation T T T T T T T T 1000 1500 2000 2500 3000 3500 4000 4500 101 Local Southern method 102 Possible local sizing inaccuracy Mobility of any DNA fragment can be affected by its sequence and by secondary and tertiary structure formation If any internal size standard fragment has anomalous mobility the Cubic Spline method may exhibit local sizing inaccuracy For example Assume that a standard fragment is close in molecular length to an unknown sample fragment Assume further that the standard fragment runs anomalously The Cubic Spline method assigns the official value to this standard fragment even though it may be slightly incorrect The size of the unknown fragment is t
76. TGTAGCGCTTAGA AAGCAACAT CGCTAATCT The G C and A T base pairs are two ui possible alleles of TCGTTGTAACGCTTAGQA this SNP site AGCAACAT T GCTAATCT SNP markers occur in the human genome at a frequency of about 1 in every 1000 bp with a total number of over 10 million SNP markers distributed evenly over the 3 billion bps of the human genome They have been shown to be responsible for differences in genetic traits susceptibility to disease and response to drug therapies SNP markers are excellent genetic markers to construct high resolution genetic maps SNP markers can be genotyped by a variety of methods Life Technologies products support the following methods Single base extension Shifted Termination Assay STA primer extension Applications SNP Some applications of SNP genotyping include Study of mutations implicated in various cancers Genetic disease research Mitochondrial DNA investigations Scrapie susceptibility in sheep Loss of heterozygosity Assess performance in food animal production Differentiate drug and non drug forms of Cannabis DNA Fragment Analysis by Capillary Electrophoresis 119 SNaPshot Multiplex System Components 120 The SNaPshot Multiplex System investigates up to ten SNP markers simultaneously by using PCR amplification then dideoxy single base extension of an unlabeled primer and then capillary electrophoresis After electrophoresis and fluorescence detection the
77. The recommended injection solvent Hi Di Formamide is highly deionized formamide formulated with a stabilizer Storage of Hi Di Formamide is important in maintaining the quality and conductivity of the solvent See Hi Di Formamide storage on page 82 For more information see Irregular signal intensity troubleshooting on page 164 Optimizing electrokinetic injection parameters 78 Electrokinetic injection parameters affect data quality run to run precision in sizing and reproducibility in the amount of sample loaded Optimize parameters to inject sufficient DNA to yield peaks of adequate height that is data with a good signal to noise ratio while maintaining the resolution and precision required by the application The Data Collection Software includes run modules with preset values for injection times and voltages that have been optimized for specific instrument polymer capillary length configurations These values are adequate for many applications However consider modifying the injection parameters if the run modules yield signal that is too strong or too weak or if the resolution is poor The maximum recommended injection time is 30 seconds and the maximum possible injection voltage is 15 kV DNA Fragment Analysis by Capillary Electrophoresis Definition of resolution Optimizing injection time Chapter 4 Optimizing Capillary Electrophoresis The resolution R of two peaks in an electropherogram is defined
78. a chemistry and the AnyDye dye set template 4 Select the dye colors to use and set the calibration peak order 70 DNA Fragment Analysis by Capillary Electrophoresis Chapter 4 Optimizing Capillary Electrophoresis a Select the dye colors to use which specifies the order number of the dye used internally by the software Note that when you deselect a dye the order number of the dye used internally by the software changes The examples below are for a 3500 Series instrument with 6 dye support but the logic applies to 4 and 5 dyes In Example 1 with all dyes selected internal order number is Blue 1 Green 2 Yellow 3 Red 4 Purple 5 Orange 6 In Example 2 with the Purple dye deselected internal order number is Blue 1 Green 2 Yellow 3 Red 4 Orange 5 the internal order number of Orange changes to 5 n Example 3 with the Blue Yellow and Purple dyes deselected internal order number is Green 1 Red 2 Orange 3 the internal order number of Green changes to 1 Red changes to 2 and Orange changes to 3 Dye Selection Example 1 1 2 3 4 5 6 E o0 NEN o0 0v Example 2 1 2 3 4 5 Internal order numbers Mem CT NNUS ENENNE o B displayed on the screen Example 3 1 2 3 ELEM US o NENNEN o S b Specify the order of the peaks in the calibration standard you are using Use the internal order number of the dye based on the dyes selected IMPORTANT The Calibration Peak Ord
79. agarose gels with ethidium bromide staining The intensity of emitted fluorescence is different for each dye and you must optimize sample concentration to account for differences in dye signal strength For example to generate signals of equal intensity you must load approximately three times as much TM NED dye labeled fragments as 6 FAM dye labeled fragments For more information see e Chapter 3 Optimizing PCR on page 55 Chapter 4 Optimizing Capillary Electrophoresis on page 67 DNA Fragment Analysis by Capillary Electrophoresis 53 54 DNA Fragment Analysis by Capillary Electrophoresis Optimizing PCR This chapter contains general information for PCR For application specific information on PCR see the application chapters later in this guide This chapter covers Safety information eies ae a n aae EAEE een 55 Isolating purifying quantifying and storing DNA 55 Handling primers oe TE s E pepe mt ahr 57 Using control DNAs ceders ee jets dank AER a mae aoe e aei re e bre I 57 Reaction volumes and plate types cc cece eee eee 58 Reagent concentrationsicn 2 2st healt ee ee ide iE te er ota 59 Preventing competing side reactions hot start PCR 0000000 ee 60 Thermal cycling parameters Veriti Thermal Cyclers 00000 60 Thermal cycling parameters 9700 Thermal Cyclers 0 0 00 cece eee 62 Optimizing thermal cycling parameters sse eee 63 Avoiding contamin
80. age 25 Do fragment sizes overlap see Compensating for overlapping fragment sizes on page 28 Will you evaluate one target per reaction singleplex or multiple targets per reaction multiplex see Singleplexing versus multiplexing on page 26 What factors affect the design of your primers see Primer design guidelines on page 29 Which dye sets are compatible with your genetic analyzer and are appropriate for the number of markers of interest see Dye sets on page 41 and Singleplexing versus multiplexing on page 26 Which size standard is appropriate for the fragment size range and dye labels of your samples see Size standards on page 42 DNA Fragment Analysis by Capillary Electrophoresis 21 DNA polymerase enzymes Overview For most applications AmpliTaq Gold DNA Polymerase is the enzyme of choice However Life Technologies supplies a number of PCR enzymes that have been optimized for specific needs as listed below Go to www lifetechnologies com for other available enzymes Note AmpFSTR AFLP and SNaPShot kits include the appropriate DNA polymerase for the application Table 1 PCR enzymes supplied by Life Technologies DNA polymerases Description AccuPrime Taq DNA Polymerase System Provides reagents for amplification of nucleic acid templates with antibody mediated hot start for improved PCR specificity over other hot start DNA polymerases Platinum anti Tag DNA polymerase antibod
81. ajor analysis issues Each primer should have 23 nt complimentary to the gDNA sequence Use 5 tails to create different length primers Add poly dGACT to generate a size difference of at least 4 to 6 nt Primers can be complimentary to the strand of the DNA if the strand is difficult to assay Always run a negative control no template DNA when evaluating a new primer Design primers 2 Prepare template by PCR of target then clean up 3 Prepare SNaPshot reactions 4 5 6 Post extension by PCR then clean up Capillary electrophoresis Analyze data Data analysis The GeneMapper Software includes a SNaPshot Default analysis method that you can use as a starting point for analysis For more For documents and publications see SNP applications on page 201 information An extensive list of publications demonstrating the utility of the SNaPshot Multiplex System is available at www lifetechnologies com snapshot For ordering information see SNaPshot Kits on page 198 DNA Fragment Analysis by Capillary Electrophoresis 123 124 DNA Fragment Analysis by Capillary Electrophoresis Fingerprinting B OVERVIEW oe veut bet uva een EE 125 m Amplified fragment length polymorphism AFLP Analysis 126 B Terminal restriction fragment length polymorphism T RFLP 131 m Bacterial Artificial Chromosome BAC fingerprinting 132 m High coverage expression
82. ak height will give similar results If the peaks are irregularly shaped or have shoulders peak heights will often give better results than peak areas Iftwo fragments differ greatly in size compare peak areas because large peaks tend to spread considerably more than small peaks For more information For documents and publications see LOH applications on page 200 For ordering information see Ordering Information on page 193 Microsatellite Instability MSI and Replication Error RER Microsatellite instability MSI describes the reduced fidelity during the replication of repetitive DNA often occurring in tumor cells It is thought to be caused by strand slippage during DNA replication due to mutations in DNA mismatch repair genes MSI leads to the appearance of multiple alleles at microsatellite loci Replication error RER is usually defined as MSI at multiple microsatellite markers or loci The appearance of numerous extra alleles at lower molecular weights in the tumor sample Figure 42 bottom panel indicates significant genomic instability 146 DNA Fragment Analysis by Capillary Electrophoresis Chapter 9 Relative Fluorescence Quantitation RFQ 9 Figure 42 Microsatellite instability identified by lower peak signal intensity in the tumor sample 7 93 sa 10 112 i 79 145 Normal OB live Tumor The technique for detecting RER involves comparing microsatellite alleles after PCR amplifi
83. alyze data using this panel A change in the fragment profile can be indicated by the absence of a peak as well as a reduction in the height of a peak when comparing different samples The following two figures are examples of typical and polymorphic AFLP reactions Figure 31 Typical electropherogram of an AFLP reaction 1000 800 200 300 400 500 AFLP com Fl 00 2004 09 03 fsa 1000 800 AFLP cam AFLP Tuil Pand ij O 200 300 400 500 DNA Fragment Analysis by Capillary Electrophoresis 129 Figure 32 Polymorphic AFLP peaks 90 130 170 20 250 230 370 40 8 8 8 JAFLP_sample_AD AFLP sample internal Panel mers 9 A B 5 amp 9 RH R 9 R pJ 30 130 170 210 250 290 330 370 410 450 450 8 88 8 lea anr Ta l Kae T al K eaa T BT T T T zll AFLP zamele DO AFLP zample interna Panel intera G9 B 34 L V g 8 8 8 AFLP sample FO AFLP sample interna Panel intermu 9 9 D S Linternal Marker Dye These peak patterns are automatically converted to a table of binary marker genotypes Figure 33 which can be exported and analyzed for similarity and generation of dendrograms using a statistical software package or other downstream analysis software for this type of clustering analysis Figure 33 AFLP genotypes in GeneMapper Software Samples Genotypes
84. alyzers 4410228 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 3730 3730xl Analyzer materials Capillary Array 50 cm 48 Capillary 3730 for Genetic Analyzers 4331250 Capillary Array 36 cm 48 Capillary 3730 for Genetic Analyzers 4331247 Capillary Array 50 cm 96 Capillary 3730xl for Genetic Analyzers 4331246 Capillary Array 36 cm 96 Capillary 3730xl for Genetic Analyzers 4331244 GeneScan Size Standards See GeneScan size standards on page 197 194 DNA Fragment Analysis by Capillary Electrophoresis Ordering Information Item Source Matrix Standard Kits See Matrix standards for spectral calibration on page 197 POP 7 Polymer 28 mL 3730 3730xl Genetic Analyzers 4363929 POP 7 Polymer 140 mL 3730 3730xl Genetic Analyzers 4335615 POP 7 Polymer 280 mL 3730 3730xl Genetic Analyzers 4363935 POP 7 Polymer 840 mL 3730 3730xl Genetic Analyzers 4335611 MicroAmp Optical 96 Well Reaction Plate N8010560 Hi Di Formamide 4311320 Reservoir Septa 4315932 Running Buffer 10x 402824 96 Well Plate Septa 4315933 3130 3130xl Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 Capillary Array 80 cm 4 Capillary 3130 Genetic Analyzers 4333465 Capillary Array 50 cm 4 Capillary 3130 Genetic Analyzers 4333466 Capillary Array 36 cm 4 Capillary 3130 Genetic Analyzers 4333464 Cap
85. and 3 and 5 RACE Purified from E coli DNA Fragment Analysis by Capillary Electrophoresis 23 Derivatives of Tth DNA polymerase Enzyme characteristics 24 Life Technologies supplies two modified forms of Thermus thermophilus Tth DNA polymerase rTth DNA Polymerase is obtained by expression of a modified form of the Tth gene in an E coli host rTth DNA Polymerase XL Extra Long provides the same features as rTth DNA Polymerase for target sequences from 5 to 40 kb An inherent 3 5 exonuclease activity allows for the correction of nucleotide misincorporations that might otherwise prematurely terminate synthesis Table 2 Enzyme characteristics Characteristics Recommended enzyme xL igh specificity AccuPrime Tag DNA Polymerase I igh sensitivity I igh fidelity I igh temperatures Multiplex PCR Amplification of low copy number template AmpliTaq Gold DNA Polymerase Platinum Pfx DNA Polymerase AmpliTaq DNA Polymerase Platinum Multiplex PCR Master Mix AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase s AmpliTaq DNA Polymerase LD for bacterial sequences High specificity at high ionic strength AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase Amplification of extra long fragments 55 kb rTth DNA Polymerase XL Pre PCR conversion to cDNA SuperScript IIl Reverse Transcriptase Extra cycles AmpliTaq9
86. and EPT trace below Examine the 1 In the Project window select a sample in the navigation pane l sample info raw E gaProject Info Raw Data EPT Data data and EPT trace S E Project 01 Sample 01 fsa Sample Information Sample 02 fsa Sample File Sample 01 fsa Sample 03 fsa Sample Name Sample 01 Sample Origin Path C Applied Status Message Analyzed File Source Disk medig Error Message 2 Check for error messages Note If this error sa message is Alert displayed at any time when you are using the software check the Info tab to determine the error There are sample s that do not meet analysis requirements Please see Error Message in the Info view of each sample Do you want to continue 3 Review the sample information Ensure that the correct analysis settings and data collection setting were used 154 DNA Fragment Analysis by Capillary Electrophoresis 4 Click the Raw Data tab Chapter 11 Troubleshooting Note The Raw Data tab is the only place in the software in which you can view Negative baselines e Run data for SQ samples The example below illustrates good quality raw data for multiplexed microsatellite data 5 Click the EPT Data tab Review the current voltage temperature and power throughout the electrophoresis run Large fluctuations in the values can result in poor quality data The e
87. and you can encounter reproducibility and contamination problems Thermal cycling parameters Veriti Thermal Cyclers AmpliTaq Gold 60 Use this profile for hot start PCR in place of labor intensive methods such as manual hot start or wax bead mediated hot start techniques The Hot start technique helps to minimize the formation of primer dimers or non specific products thereby increasing specificity and sensitivity of PCR This profile specifies a pre PCR heat step for activation of AmpliTaq Gold DNA Polymerase DNA Fragment Analysis by Capillary Electrophoresis General PCR Time release PCR Touchdown PCR Chapter 3 Optimizing PCR Use this profile for standard PCR Use this profile with AmpliTaq Gold DNA Polymerase This method minimizes the pre PCR activation step and adds a minimum of 10 additional cycles allowing for slow activation of the enzyme during cycling This provides a simple method where polymerase activity increases more slowly as product accumulates improving specificity Use this profile if the optimal annealing temperature is not known This method incrementally decreases the annealing temperature in early cycles to maximize the yield of specific products DNA Fragment Analysis by Capillary Electrophoresis 61 Thermal cycling parameters 9700 Thermal Cyclers AmpliTa q_Gold Use this profile for hot start PCR in place of labor intensive methods such as manual hot start or wax bead mediated ho
88. andards ss eo eU bes te debe east eda eas 197 m Matrix standards for spectral calibration eee 197 B Installation standards 2 ccc ccc cece eee eee 197 a Reagent kits soe ESSERE ERU loreal adi diy Bod Sood 198 m Other user supplied materials 198 Thermal cyclers and accessories Item Source Veriti 96 Well Thermal Cycler 4375786 GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 Silver 96 Well Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 MicroAmp Autoclaved Reaction Tubes with Caps 0 2 mL N8010612 GeneAmp Autoclaved Thin walled Reaction Tubes with Domed Caps 2000 tubes N8010537 GeneAmp Autoclaved Thin walled Reaction Tubes with Domed Caps 1000 tubes N8010611 MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Cap Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 DNA Fragment Analysis by Capillary Electrophoresis 193 Genetic analyzers and consumables Itemt Source Applied Biosystems 3500 or 3500xL Genetic Analyzer Contact your local Life Technologies sales representative
89. apillary Electrophoresis 83 Understanding spatial calibration A spatial calibration maps each capillary location to the CCD camera which detects the signal for each capillary A f T fl Capillaries 8 Jr mt Bc s k Ee Era aora RAT e Fase a ea at ee Ra eee ee ed Ep Eau 12 0 2 40 6 8 100 120 140 160 180 200 220 240 250 280 300 320 340 350 380 400 420 440 460 480 5 8 S000 12000 Good spatial calibration Bad spatial calibration Spatial calibration maximizes data quality and accuracy Refer to the instrument user guide for instructions on performing a spatial calibration See Instrument documentation on page 199 for document part numbers Perform a spatial calibration when you Install or replace a capillary array Temporarily remove the capillary array from the detection block Move the instrument Open the detection block Understanding spectral calibration IMPORTANT Always visually examine the data generated by a spectral calibration Accepting a calibration with poor data will yield inaccurate results when you run samples For information on creating a custom dye set for spectral calibration see the instrument sections earlier in this chapter 84 DNA Fragment Analysis by Capillary Electrophoresis Chapter 4 Optimizing Capillary Electrophoresis Spectral A spectral calibration or matrix on 310 instruments allows the software to distinguish calibration betwe
90. applied biosystems by ife technologie DNA FRAGMENT ANALYSIS by Capillary Electrophoresis 0 technologies USER GUIDE applied biosystems oy HE technologies DNA Fragment Analysis by Capillary Electrophoresis Publication Number 4474504 Rev A Revision Date September 2012 6 technologies For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF NOTICE TO PURCHASER LIMITED USE LABEL LICENSE Research Use Only The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for
91. aq Gold DNA Polymerase is recommended for each 100 uL reaction volume Note To avoid the inaccuracies involved in pipetting 0 5 uL amounts of enzyme into each reaction prepare a fresh master mix of reagents and add the enzyme Preventing competing side reactions hot start PCR When to use Limitations and alternatives Consider using the hot start technique whenever you need to improve the specificity and sensitivity of your PCR amplifications Loss of specificity and sensitivity are often caused by competing side reactions which usually occur during the pre PCR setup period A common competing side reaction involves the amplification of non target sequences in background DNA due to mispriming or to primer oligomerization The hot start technique is cumbersome If you have high throughput needs switching to AmpliTaq Gold DNA Polymerase will give the same benefits as performing the hot start technique without the need for using wax barriers or opening reaction tubes If you are already using AmpliTaq Gold DNA Polymerase performing the hot start technique will not improve the specificity and sensitivity of PCR amplification Components necessary for amplification must be kept separately so that critical reactants do not mix until reaching a temperature sufficiently high to suppress primer self annealing or annealing to non target sequences Note Although manual hot start PCR can increase specificity and yield it is inconvenient
92. at the wavelength of maximum absorption Quantum yield The probability that its excited state will emit a photon as it decays back to the ground state The ability of the instrument to detect a dye signal depends upon The absorption efficiency of the dye at the wavelengths of light emitted by the laser Thelaser light source Thequantum yield of the dye The dye concentration The emission and absorption wavelengths of a dye depend upon The chemical structure of the dye The physical environment including Buffer pH and concentration Polymer composition Whether the DNA it is attached to is single or double stranded Although altered by the physical environment the wavelengths of maximum emission and absorption for each dye always lie within a small wavelength range DNA Fragment Analysis by Capillary Electrophoresis 37 Emission and absorption excitation wavelengths and relative intensities 38 The maximum fluorescence absorption and emission wavelengths are listed below for Life Technologies NHS esters dye phosphoramidites and F dNTP based dyes The actual maximum absorption and emission wavelengths may differ from the listed values because of the influence of the physical environment upon the dye The intensity of emitted fluorescence is different for each dye and you must optimize sample concentration to account for differences in dye signal strength Examples e 6 FAM dye emits a stronge
93. aterfall most common on 310 instruments Baseline waterfall g 3 3090 000 7000 6000 5000 4000 3000 2000 i e Contamination from marker pen ink if you used a marker to label the plate or the heat seal Prepare new plate 178 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible Cause Action continued Constant elevated signal in raw data e Waterfall most common on 310 instruments Contamination from water used to make buffer wash reservoirs septa for sample injection for any sample prep steps or for water wash Use fresh water Instrument contamination Improperly filled leaky connections tubing or polymer block Refer to the instrument user guide for troubleshooting information Spectral matrix calibration issue Use correct matrix standard see Dye sets and matrix standards on page 41 Specify the correct dye set in the instrument protocol Ensure the correct dye set was selected in spectral calibration Apply the correct matrix file 310 instruments only Polymer on instrument gt 7 days polymer degraded or precipitated Perform warm water washles and replace polymer If the problem persists replace the array Arcing electronic noise Remove bubbles Refer to the instrument user guide for information Noisy baseli
94. ation lt 0 eee eene 64 Safety information IMPORTANT For every chemical read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Isolating purifying quantifying and storing DNA Isolating DNA DNA isolation methods depend on your starting DNA source Refer to guidelines for your application for information on isolating DNA IMPORTANT DO NOT FREEZE BLOOD SAMPLES before DNA isolation Freezing can lyse red blood cells and increase the concentration of PCR inhibitors in DNA samples DNA Fragment Analysis by Capillary Electrophoresis 55 Purifying DNA Quantifying DNA Storing prepared DNA before or after PCR 56 The quality accuracy and amplified length of a DNA fragment can be significantly affected by characteristics of the sample itself and the method used for purification IMPORTANT The success of AFLP analysis is particularly dependent upon the quality of DNA Select a method based on the sample source or tissue type how it was obtained from its source and how it was handled or stored before purification Go to www lifetechnologies com for the latest information on DNA purification For PCR with custom primers optimize DNA concentration for your application Concentration may range from 10 to 100 ng of purified DNA per rea
95. available on our website Before using the downloaded run modules adjust as described below Further optimization may be necessary 50 cm array Decrease run voltage to 8000 volts Increase run time to 6200 seconds DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design GeneScan 350 Range 35 to 350 bp under denaturing conditions ROX Size Standard GeneScan ROX 350 denatured fragment lengths nt 12 fragments 35 139 250 50 150 300 75 160 340 100 200 350 Do not use this fragment for sizing See Peaks not used for sizing on page 43 for information Only one strand of the double stranded DNA fragments in this size standard is labeled The unlabeled strand does not interfere with peak detection of the labeled strand when run under denaturing conditions Figure 12 GeneScan 350 Size Standard run under denaturing conditions GeneScan 400HD This size standard uses ROX dye The high density of marker bands in this standard ROX Size makes it particularly useful for microsatellite analysis Standard Range 50 to 400 bp under denaturing conditions GeneScan ROX 4OOHD denatured fragment lengths nt 21 fragments 50 160 260 360 60 180 280 380 90 190 290 400 100 200 300 120 220 320 150 240 340 Only one strand of the double stranded DNA fragments in this size standard is
96. avoid mobility shifts that interfere with accurate interpretation of data DNA extraction and purification Because AFLP analysis requires only a small amount of DNA 50 to 500 ng ideally 10 to 100 ng DNA purification is critical We recommend the following kits for extracting DNA for AFLP analysis e Plant Analysis DNAzol Reagent or PureLink Genomic Plant DNA Purification Kit Microbial Analysis PureLink Genomic DNA Mini Kit DNA Fragment Analysis by Capillary Electrophoresis 127 Workflow 128 Restriction In AFLP experiments on genomes of unknown content determine whether or not your genomic DNA restricts properly with EcoRI and Msel enzymes In general the Regular Plant Genome Kit modules should produce quality genetic fingerprints with genomes of 5x108 to 6X10 base pairs and the Small Plant Genome Kit modules with genomes of 5x107 to 5X108 base pairs Empirical guidelines suggest that if the G C content of the genome is 76576 Msel will not give a significant number of fragments Optimal results are obtained with Msel when the G4C content is 5076 EcoRI also tends to produce more fragments in G C poor genomes In cases where an organism s G C content is unknown the effectiveness of the restriction enzymes must be determined empirically Primers For the selective amplification step the primers that target the EcoRI A binding site are fluorescently labeled at the 5 end The primers that target the M
97. called pull ups are caused by too little subtraction Bleedthrough peaks are small peaks of one color lying directly under a large peak of another color even though there is no PCR product corresponding to the smaller peak Elevated interpeak baseline is caused by too much subtraction The table on the next page contains examples of the symptoms listed above DNA Fragment Analysis by Capillary Electrophoresis 191 Symptom Possible Cause Action Pull up peaks too little matrix subtraction The matrix was made with the wrong Create a new matrix with the correct dye and dyes or filter set filter set See Dye sets on page 41 The signal from a large peak is off scale Keep peak heights between approximately 150 because of sample overloading In the and 4000 RFU If sample data is off scale do raw data the peak showing pull up is one of the following off scale e Rerun the samples using a shorter injection time Dilute and rerun the samples Create a new matrix with the correct dye and filter set See Dye sets on page 41 Elevated interpeak baseline too much matrix subtraction Too much matrix subtraction Create a new matrix 192 DNA Fragment Analysis by Capillary Electrophoresis Ordering Information m Thermal cyclers and accessories 0 eens 193 B Genetic analyzers and consumables 0 c cece eens 194 E GeneScan size st
98. cation in normal and tumor samples from the same host You calculate a raw RER score using an algebraic formula that quantifies the relative strength of the stutter peaks in the two samples after normalizing for differences in PCR efficiency While both microsatellite instability and loss of heterozygosity are indicative of cancerous tissue if an electropherogram shows RER at a given marker location an LOH calculation for that allele region is complicated or even invalid Canzian et al 1996 We do not recommend LOH calculations in regions that show clear signs of RER DNA Fragment Analysis by Capillary Electrophoresis 147 148 DNA Fragment Analysis by Capillary Electrophoresis 0 Additional Applications E DNA methylation se SE a E a Re REP ERU EVER eS eee 149 DNA methylation The study of methylation epigenetics is emerging as an important component of cancer research In a typical assay to detect methylation bisulfite treatment of DNA deaminates non methylated cytosine and converts it to uracil while methylated cytosine remains unreactive The subsequent step of PCR amplification converts uracil bases to thymine Use the SNaPshot system to quantitatively detect the base differences in treated and untreated samples to learn the methylation status of the samples For more information see Methylation applications on page 200 DNA Fragment Analysis by Capillary Electrophoresis 149 150 DNA Fragment A
99. ch as run temperature voltage or the denaturing ability of the separation matrix s Polymer type POP 49 POP 6 POP 7 and concentration e Capillary length 22 cm 36 cm or 50 cm Instrument model due to differences in instrument configuration GeneMapper Software sizing methods Least Squares method 100 The sizing methods available in classic and advanced modes in the analysis method of the GeneMapper Software are e Least Squares Cubic Spline Interpolation Local Southern e Global Southern Global methods which generate the best fit curve from all matched fragments in the size standard are less affected than local methods by anomalies in the run times of single size standard fragments Does not normalize capillary to capillary Local methods which generate the best fit curve from nearby size standard data points are less affected by changes in the electrophoresis conditions or in the analysis range A change in the analysis range changes the subset of size standard fragments that is available for generating the sizing curve Normalizes capillary to capillary Advantages Both Least Squares methods 2nd Order and 3rd Order use regression analysis to build a best fit sizing curve This curve compensates for any fragments that may run anomalously As a result this method normally results in the least amount of deviation for all the fragments including the size standards and the samples Depending on whet
100. cifications 68 throughput 69 DNA Fragment Analysis by Capillary Electrophoresis Index A AccuPrime Taq DNA Polymerase 22 24 AccuPrime Taq DNA Polymerase High Fidelity 22 AFLP advantages 126 analysis method GeneMapper Software 129 applications 127 data analysis 129 example data 129 experiment and primer design recommendations 127 genotypes in GeneMapper Software 130 instrument and consumable recommendations 127 overview 125 PCR kits 198 principle 126 restriction enzymes 128 workflow 128 agarose gel using for troubleshooting 157 ambient temperature problems caused by 160 AmpiTaq Gold DNA Polymerase 23 amplification See also PCR non specific 32 post amplification manipulations 33 selective 31 amplified fragment length polymorphism See AFLP AmpliTaq DNA Polymerase 22 24 AmpliTaq DNA Polymerase LD 22 24 AmpliTaq Gold DNA Polymerase 24 analysis software 89 aneuploidy relative fluorescence application 17 animal breeding microsatellite application 16 animal typing microsatellite application 16 annealing temperature optimizing 64 PCR parameters for unknown 61 63 annealing factors affecting 30 association studies 110 autosampler problems caused by misalignment 161 211 B BAC fingerprinting applications 133 data analysis 73 90 159 experiment and primer design recommendations 134 instrument and consumable recommendations 134 overview 132 principle 133 workflow 135 bacterial artificial chromosom
101. consistent amplification Doyle and Doyle 1993 In DNA amplification primers and PCR master mixes should be tested for robustness and consistency when amplifying ISSR targets in both species Subsequently thermal cycling conditions can be refined with particular focus on primer annealing temperature and primer annealing time For additional information on optimization refer to ISSR Genotyping of Endangered Plants Using an Optimized Workflow on page 200 DNA Extraction Detection 3500 Series GeneMapper AmpliTag Gold 360 Genetic Analyzer v4 1 Software CTAB Master Mix l GeneScan 1200 LIZ Modified AFLP Size Standard Analysis Method In the GeneMapper Software In the Panel Manager create a panel for each dye color primer with bins centered at whole base pairs one base pair wide covering the entire range of 80 to 1200 bp Figure 37 In the GeneMapper Manager modify the AFLP Analysis Method Figure 38 This method detects peaks above a minimum peak height as an allele and applies a binary label of either 1 or 0 for the presence of a peak in a particular bin Figure 37 Creating multiple ISSR bins and example of multiple bins centered at whole base pairs for the blue marker in an ISSR Pane lw x IE L LRR See eee D s DC E DNA Fragment Analysis by Capillary Electrophoresis 139 140 Figure 38 Example of the Allele tab settings for an ISSR analysis me
102. converted into digital data then the data is stored in a file format compatible with an analysis software application DNA Fragment Analysis by Capillary Electrophoresis Fragment analysis workflow Chapter 1 Introduction to Fragment Analysis Phase Technology Life Technologies products used 1 Isolate DNA Depends on sample source DNA isolation methods depend on your starting DNA source and application Refer to guidelines for your application for information on isolating DNA 2 Purify DNA Depends on sample source Go to www lifetechnologies com for advice on the and application appropriate product to use 3 Quantify DNA Dye labeling and Qubit Fluorometer and Quantitation Kit go to fluorometric detection www lifetechnologies com qubit 4 PCR Dye labeling and Veriti Thermal Cycler amplification amplification of fragments using a thermal cycler 96 well 384 well GeneAmp PCR System 9700 Dual 96 well Dual 384 well Auto Lid Dual 384 well e 2720 Thermal Cycler 5 Capillary electrophoresis Separation of fragments based on size using a genetic analyzer e 3500 3500xL Genetic Analyzer 3500 Series instrument e 3730 3730xl Genetic Analyzer 3730 Series instrument e 3130 3130xl Genetic Analyzer 3130 Series instrument e 310 Genetic Analyzer 310 instrument 6 Data analysis Sizing and optional s GeneMapper Software genotyping Sizing Peak Scanner Software available free of c
103. ction It is almost always necessary to dilute PCR amplification products before adding them to the sample tube Typically the required dilution is 1 3 to 1 80 PCR product to distilled deionized water or Hi Di Formamide Begin by optimizing PCR run conditions for your specific application Then run a dilution series on your instrument to determine the optimal dilution Alternatively run 1 uL of PCR product on a mini gel If after ethidium bromide staining the product signal is visible but not oversaturated try a 1 10 dilution After determining the optimal dilution ratio you can use the same dilutions for subsequent analyses because PCR yields should be fairly consistent Any changes to the PCR conditions or the primer design may require different dilutions Note Different dyes emit different fluorescent intensities Therefore PCR product concentrations may need optimization depending on relative fluorescence intensity during electrophoresis See Emission and absorption excitation wavelengths and relative intensities on page 38 Store the prepared samples at 20 C to 4 C until you perform capillary electrophoresis DNA Fragment Analysis by Capillary Electrophoresis Handling primers Reconstituting and diluting primers Quantifying primers Storing primers Chapter 3 Optimizing PCR Primers are commonly shipped in a lyophilized state The units of a lyophilized primer are given as a mass in picomoles To c
104. d Support References Glossary 14 DNA Fragment Analysis by Capillary Electrophoresis Introduction to Fragment Analysis m Fragment analysis versus sequencing what is the difference 15 B What can I do with fragment analysis 0 ees 16 m What is capillary electrophoresis 0 66 c cece cece eens 18 E Fragment analysis workflow seeeeee eee teen ees 19 Fragment analysis versus sequencing what is the difference Fragment analysis Fragment analysis using Life Technologies products involves Labeling fragments with fluorescent dyes Multiple different colored fluorescent dyes can be detected in one sample One of the dye colors is used for a labeled size standard present in each sample The size standard is used to extrapolate the base pair sizes of the sample product peaks Amplifying the labeled fragments using polymerase chain reaction PCR on a thermal cycler Separating the fragments by size using capillary electrophoresis Analyzing the data using software to determine Size The analysis software uses the size standard in each sample to create a standard curve for each sample It then determines the relative size of each dye labeled fragment in the sample by comparing fragments with the standard curve for that specific sample Genotype The analysis software assigns allele calls based on user defined makers loci Figure 1 Fragment analysis fluorescent
105. d to a porous substrate in order to separate molecules by size or shape For example nucleic acids like DNA and RNA are negatively charged and are thus attracted to a positive charge and will move toward it through a matrix like a polymer or agarose gel The strength of the electrical field and charge on the molecules and the size of the pores in the matrix determine the speed at which different molecules in the starting mixture migrate In general smaller molecules migrate faster than larger molecules because they are less obstructed as they travel through the matrix The intensity of emitted light fluorescence as a function of the wavelength of the emitted light DNA Fragment Analysis by Capillary Electrophoresis 207 excitation efficiency excitation spectrum filter set fingerprint genetic mapping Linkage mapping ISSR PCR locus marker microsatellites minisatellites panel phylogenetic studies polymerase polymorphic locus 208 The probability that it will absorb light of a certain wavelength as a percentage of the probability of absorption at the wavelength of maximum absorption The intensity of emitted light as a function of the wavelength of the exciting light Physical filters that separate dye signals in older gel based capillary electrophoresis instruments Newer Life Technologies instruments use a diffraction grating A characteristic pattern of peaks or bands after an amplification or diges
106. dard fragments run anomalously DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software This is how the Local Southern method works 1 The fitting constants of the curve are calculated for each group of three neighboring points on the standard A separate curve is created for each set of three points 2 Acurve is then created by using three standard points two points below and one point above the fragment and a fragment size is determined 3 Another curve is created by looking at an additional set of three points one point below and two points above the fragment and another value is assigned 4 The two size values are averaged to determine the unknown fragment length Global Southern This method is similar to the Least Squares method in that it compensates for standard method fragments that may run anomalously The method creates a best fit line through all the available points and then uses values found on that line to calculate the fragment values Global Southern sizing curve Best Fit 2nd Order Curve AO 9 71623E 01 Al 1 220838E 01 A2 4 856885E 06 Size Calling Curve Global Southern Method T T Global Southern method equations Equation Description Attempts to describe the reciprocal L c m m0 4 LO relationship between the mobility m and the length LO of the standard fragments The fitting constants LO
107. database of known species Applications Examine microbial community structure and community dynamics in response to changes in different environmental parameters or to study bacterial populations in natural habitats e Study of complex microbial communities in diverse environments such as soil Derakshani and Lukow et al marine and activated sludge systems Eschenhagen and Schuppler et al e Characterize oral bacterial flora in saliva in healthy subjects versus patients with periodontitis Sakamoto and Takeuchi et al Preliminary screening of microorganisms before analysis using Applied Biosystems MicroSEQ Microbial identification kits Instrument and This is a Life Technologies demonstrated protocol consumable Thermal cycler Veriti GeneAmp 9700 2720 recommendations Genetic analyzer 3500 Series 3730 Series 3130 Series and 310 instruments Polymer see Run modules on page 69 for the polymer and capillary array length combinations supported on each instrument e GeneScan 600 LIZ Size Standard e DS 33 Matrix Standard Dye Set G IMPORTANT Throughout a set of experiments use all the same equipment run parameters polymers dyes and so on Consistent conditions are required to avoid mobility shifts that interfere with accurate interpretation of data DNA Fragment Analysis by Capillary Electrophoresis 131 Experiment and primer design recommendations Workflow Data analysis For mo
108. dicates potential loss of heterozygosity LOH in the sample DNA Fragment Analysis by Capillary Electrophoresis 143 Applications Figure 41 Example electropherogram of healthy and tumor samples MI P P9 MA P ee va gt e Tes ss Healthy Tumor Screening for loss of heterozygosity LOH using microsatellites or Single Nucleotide Polymorphisms SNPs Aneuploidy assays Detection of large chromosomal deletions Multiplex ligation dependant probe amplification MLPA Experiment and primer design recommendations Recommendations Minimizing signal intensity variation 144 Do not use internally labeled F dNTP labeled fragments in quantitative experiments Variations in the per fragment number of labeled nucleotides and the increased peak spreading with this method make relative quantitation unreliable For more information see Microsatellite Analysis on page 107 and Amplified fragment length polymorphism AFLP Analysis on page 126 To minimize variations consider the ionic strength of samples and consumables The amount of DNA injected is inversely proportional to the ionic strength of the solution Samples high in salt result in poor injections PCR reactions vary in efficiency therefore some reactions may result in higher ionic concentration post amplification Conductivity of the solvent used for injection will affect the sample injection and can cause variation in
109. dyes NHS esters Post synthesis 5 end labeling of oligonucleotides containing a 5 NED t TAMRA t ROX RENIBUS SNaPshot Kit dyes dR110 dR 6G dTAMRA t dROX Phosphoramidite Preparing custom 5 end labeled primers directly on any Life 6 FAM HEX TET reagents Technologies DNA synthesizer8 NED t vic 9 PET t FIdNTPs Simple internal fluorescent labeling of multiple nucleotides during R6G R110 ROX TAMRA PCR amplification Labeled primersin Microsatellite and human identification applications 5 FAM TT JOE reagent kits 6 FAM HEX TET NED t VIC t PET t Labeled size Generating the sizing curve to size unknown sample fragments TAMRA ROX LIZ standard T NED VIC and PET dye labeled primers are available only in kits or through the Life Technologies Custom Oligo Service Contact your Life Technologies representative or visit our website for information on how to order custom labeled oligonucleotides T Matrix standards for spectral calibration available for the 310 instrument only For information about synthesizing labeled oligonucleotides contact your Life Technologies representative t5 FAM and JOE are available only as labeled primers in certain reagent kits Multicomponent analysis with fluorescent dyes 36 Fluorescent dye labeling enables you to analyze multiple independent markers loci in the same capillary injection by using diffe
110. e BAC DNA purification kits that are used Enzymatic digestion and SNaPshot reagent labeling can be performed in one tube or in separate reactions DNA Fragment Analysis by Capillary Electrophoresis Chapter 8 Fingerprinting 8 Workflow BAC DNA growth and purification Restriction Endonuclease Digestion Y 20 MU J ABI Pasu E EN 3 end labeling on restricted Mim et L C fragments using SNaPshot chemistry Size exclusion and signal detection on CE instrument like the 3730 3730x Peak detection and sizing using MILK i hi d aah GeneMappar data analysis Contig construction 1 Selective bacterial growth of single colonies 2 BAC purification by restriction endonuclease digestion 3 Restriction endonuclease digestion of the BAC clones with several different enzymes 4 SNaPshot reagent labeling of fragments The dye labeled primers are bound to the BAC fragments based on the overhangs left by the restriction enzymes see Table 19 on page 133 Post extension clean up of the clones not shown in diagram Capillary electrophoresis Data analysis o m Ov pm Contig construction Data analysis See BAC applications on page 200 DNA Fragment Analysis by Capillary Electrophoresis 135 For more information For documents and publications see BAC applications on page 200 For ordering information see Ordering Information on page 193 High coverage expression profiling
111. e See BAC fingerprint ing baseline troubleshooting 178 base pairing energies 30 31 bleed through peaks See pull up peaks breeding 110 C cancer progression analysis 110 capillary array for each instrument 68 problems caused by degraded or clogged 160 capillary electrophoresis See also 310 instrument 3130 Series instrument 3500 Series instrument 3730 Series instrument control DNA 57 definition 18 factors affecting 82 injection time optimizing 79 injection voltage optimizing 80 optimizing 67 optimizing conditions 80 polymers 83 run modules 46 run time optimizing 81 run voltage optimizing 81 signal intensity 77 spatial calibration 84 spectral calibration 84 troubleshooting 180 CE See capillary electrophoresis CEPH 1347 02 Control DNA 57 157 CNV relative fluorescence application 17 concentration DNA template 59 dNTP 59 212 enzyme 60 Mg ion 59 sample 158 controls CEPH 1347 02 Control DNA 157 control DNA using 57 DNA template 157 process 157 recommendation for frequency 69 156 running to isolate a problem 156 Copy Number Variation See CNV custom primer design 29 D data checking quality 104 153 electropherogram quality requirements 104 troubleshooting 153 data analysis GeneMapper Software 89 Peak Scanner Software 92 desalting 190 dinucleotide repeats 115 DNA methylation 149 DNA polymerase enzymes 3 A addition 33 characteristics 24 recommended 22 DNA template concentration 59 con
112. e and the GeneMapper Software are installed on different computers move or copy the files to another computer that has GeneMapper Software installed To perform analysis you manually apply the analysis parameters to the fragment analysis files in the GeneMapper Software GeneMapper Software features Feature Description Autoanalysis Yes with the corresponding Data Collection Software version Applications Amplified fragment length polymorphism AFLP loss of heterozygosity LOH microsatellite genotyping and SNaPshot genotyping Analysis of large fragments 1200 bp BAC fingerprinting genetic fingerprinting multilocus variant analysis MLVA Fragile X assays biodefense T B cell clonality assay bird sex identification microsatellites VNTRs T RFLP Regulatory Security and audit features to help users meet 21 CFR Part 11 requirements compliance Report Report Manager tools for customized report generation e Customization of the project auto saving frequency Analysis Definition of a linearity range in the analysis methods Process Quality Values PQVs for automated evaluation Sizing methods Least Squares Cubic Spline Local Southern and Global Southern Instrument software e Supports data generated on 3500 Series 3730 Series 3130 Series and 310 instruments e GeneMapper Software v4 1 and later includes the ability to record and reapply the Size Standard Normalization
113. e intensities Therefore concentrations of PCR products may have to be increased or decreased depending on relative fluorescence intensity during electrophoresis See Emission and absorption excitation wavelengths and relative intensities on page 38 Life Technologies genetic analyzers can convert a limited range of fluorescence signal into digital values For optimal results ensure the signal intensities are within the ranges listed below Table 16 Signal intensity ranges and fluorescence saturation Recommended signal Instrument intensity range Fluorescence saturation 3500 Series 175 10 000 RFU 30 000 RFU 3730 Series 150 10 000 RFU 30 000 RFU 3130 Series 150 4 000 RFU 8000 RFU 310 150 4000 RFU 8000 RFU The intensity of size standard peaks should be 30 to 100 of the intensity of sample peaks Dilute samples before preparing capillary electrophoresis reactions to balance the signal intensities In the example below the undiluted sample yields the correct size standard to peak intensity ratio 3888883 88 Size standard peaks should be 30 to 10096 of the intensity of sample peaks 70 oo 50 kad oo 329 240 160 90 8 59555855 DNA Fragment Analysis by Capillary Electrophoresis 77 If signal intensity is above the detection range If signal intensity is below the detection range Minimizing signal intensity variation When the signal intensity of a peak is t
114. e signal strength with noisy data 179 Symptom See page Low or fluctuating current 180 Drop off of current signal 181 Current too high 180 Instrument has stopped running and red light is on There 181 are black marks inside the instrument Symptom See page No sample plot is displayed for a sample with the error No 183 sizing data Size Match Editor does not display peak data 183 Missing size standard peaks 183 Smaller size standard peaks are not labeled 184 Larger size standard peaks are not present in trace 184 Extra peaks in size standard trace 185 Sizing failures occur in a regular pattern the same wells 186 fail repeatedly Noise peaks are detected as size standard peaks 186 Size call inaccurate for known DNA sample 186 Symptom See page GeneMapper Software error message 187 Error Message The bin set in the analysis method does not 187 match the panel used for analysis al label or alleles are not falling within bins 188 Allele not labeled 188 DNA Fragment Analysis by Capillary Electrophoresis 163 Symptom See page Data not sorted by name 188 When adding samples to a project the expected data files 188 are not listed in the Add Samples to Project dialog box Genotypes tab is grayed 189 Two peaks do not separate and are detected as one peak 189 Irregular signal intensity troubleshooting Symptom Possible cause Action No signal or low signal
115. e size standard peaks from all injections or display the sizing curve for each sample file e Verify that all defined size standard peaks are present in the Size Match Editor Variable run conditions can occasionally cause size standard peaks not to be detected for example if a run is too fast too slow or if the signal intensity of some of the peaks is too low e Use an Analysis Range that includes all the scans or data points where size standard peaks occur in the raw data of each sample To verify check the analysis method in the GeneMapper Manager DNA Fragment Analysis by Capillary Electrophoresis Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software Autoanalysis and The GeneMapper Software provides two analysis options manual analysis GeneMapper Software only Autoanalysis The software applies an analysis method size standard and optionally panel to the fragment analysis files immediately after Data Collection Software collects the data from the instrument The analysis settings are saved in the GeneMapper Software project You can review the analyzed data using GeneMapper Software e Manual Analysis You obtain the fragment analysis files from the computer connected to the instrument If the Data Collection Software and the GeneMapper Software are installed on the same computer you can import the data files into the GeneMapper Software If the Data Collection Softwar
116. e the figure below Apex Increasingly Increasingly positive slope N negative slope D Baseline 9 9 Note the following e For typical or symmetrical peaks use a value of zero e For asymmetrical peaks select values other than zero to better reflect the beginning and end points Avalue of zero does not affect the sizing accuracy or precision of an asymmetrical peak Note The size of a detected peak is the calculated apex between the start and end points of a peak Peak size does not change based on start and end settings To move the Then Example Start point of a peak closer Change the Slope Threshold for Peak Start to its apex value from zero to a positive number End point of a peak closer to Change the Slope Threshold for Peak End i its apex value to a more negative number DNA Fragment Analysis by Capillary Electrophoresis 97 How the GeneMapper Software performs sizing Size standard definitions Step 1 Size matching 98 The GeneMapper Software and the Peak Scanner Software use settings in the analysis method to size samples This section provides a brief description of the sizing process For more information see the GeneMapper Software Reference and Troubleshooting Guide Pub no 4403673 During sizing the software compares the size of observed fragments for the size standard in the sample to the expected fragment sizes listed in the size standard
117. each instrument IMPORTANT We recommend using only Life Technologies dyes Life Technologies provides calibration reagents that have been optimized for our dye sets Non Life Technologies dyes or mixed isomers of dyes have variable emission spectra and require a spectral calibration generated for the specific dyes to correct for the spectral overlap between the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure the dye labels do not affect PCR efficiency The software assumes the following dye color order blue green yellow red orange 1 In the navigation pane of the Data Collection Software click Z GA Instruments gt amp 1ga3730 or ga3130 gt Protocol Manager 2 In the Instrument Protocols pane click New The Protocol Editor opens 3 In the Protocol Editor create a spectral protocol for the Any4Dye or Any5Dye dye set specifying the appropriate protocol parameters 4 Create a Spectral Plate Record using the newly created Spectral Instrument Protocol 5 Perform a spectral calibration DNA Fragment Analysis by Capillary Electrophoresis 73 6 Set the custom dye set calibration as the active spectral calibration a In the tree pane of the Data Collection software click Z GA Instruments gt ga3130x1E or ga3130 gt CJ instrument name gt i Spectral Viewer b In the Dye Set drop down list select the custom dye set c In the L
118. eaks with a distinct pattern DNA Fragment Analysis by Capillary Electrophoresis 85 Evaluating the calibration results 86 Dye set and matrix standards for spectral calibration The table below lists the appropriate dye set and matrix calibration standard combinations for each instrument For the dye sets for applications see Table 8 on page 41 Table 18 Matrix standards see page 197 for part numbers Matrix 3730 3130 standard pye set 9908 3730xl 3130xl Mas DS 33 65 Yes Yes Yes Yes DS 02 E5 Yes Yes Yes Yes DS 32 F Yes Yes Yes Yes DS 30 D Yes Yes Yes Yes DS 31 D Yes Yes Yes Yes DS 34 C No No No Yes AnyDye Custom dye Yes Yes Yes No set you create t Because of the close proximity of capillaries on the 96 capillary 3730xl instrument we recommend using the 48 capillary 3730 instrument for fragment analysis For best results use the G5 dye set with reduced cross talk RCT configuration Use the following criteria to evaluate the data Q Value or Quality Value Measures the consistency between the final matrix and the data from which it was computed A Q value of 1 0 indicates high consistency and that no pull up or pull down peaks were detected Condition Number Represents the amount of overlap between the dye peaks in the emission spectra of the dyes in a dye set A Condition Number of 1 0 lowest possible value indicates there is no overlap in a dye set The condition number
119. eased PCR specificity Reduced PCR yield DNA Fragment Analysis by Capillary Electrophoresis 63 Guidelines The following table summarizes the effects of modifying temperature control parameters on PCR performance Change in thermal cycling parameter Effect on PCR performance Increase denaturation temperatures up to 96 C e Can be necessary to allow denaturation especially with G C rich templates e Can also cause template degradation by depurination Decrease annealing temperatures Can increase yield but can reduce specificity Increase annealing temperatures Set the denaturation annealing and extension step to at least e 15 seconds preferably 30 seconds with the GeneAmp PCR System 9700 e 45 seconds using thin walled tubes with the DNA Thermal Cycler Increases specificity but can reduce yield Allows samples to reach thermal equilibrium at each stage Use the autoextension or AutoX function of a thermal cycler to allow longer extension times in later cyclest Increases yield by allowing complete extension of PCR product in later cycles t For most applications an extension temperature of 72 C is effective and rarely requires optimization In the two temperature PCR process the combined annealing extension step temperature should range from 60 to 70 C Avoiding contamination PCR setup work area 64 PCR protocols are extremely sensitive to contaminants in t
120. ece cece eee eee 123 WOrkilOWs DET 123 DataanalysiS esce i v ue PP oe Bethe EG e ney eed gy i RU Hes RR oy eee 123 FOrmore information eerren e Eus Meta Te RA ae eater me A ed 123 DNA Fragment Analysis by Capillary Electrophoresis Contents m CHAPTERS Fingerprinting c e eee e eee eee 125 vl c 125 Amplified fragment length polymorphism AFLP Analysis 000 cece cece eee eaee 126 Principle of the analysis nannu 00 cece eee eee eee eee eae 126 Advantages is eeii TT 126 APPC ONS rs ocu L See Get eee eU cee nee Eau MET 127 Instrument and consumable recommendations cece eee eee eee eee ee 127 Experiment and primer design recommendations eee eee 127 Workflow sid thie wed b ever pie e eiue teer dee bile satis Pale peau 128 DataraMalySisSiens S1 baa dd Rr pp EL ua aeo m E o oi AR a A Lid 129 For Iriore inforrmatl ri aote hn nete eat b pA be bL Ode ac a 130 Terminal restriction fragment length polymorphism T RFLP 0 0 cece eee 131 OVERVIEW unie a otia hanks eke baa edad A dae ee raed 131 Principle of the analysis 02000 e eens 131 Applications en L Shee tot ta a eroe eese e ee ee RR ll ed 131 Instrument and consumable recommendations cece eee cece eee eee 131 Experiment and primer design recommendations cece eee e ee eee 132 Workflow os ahead ss td om seals oe ee ee Ged Db nee kamen ta xn e eR 132 Data analysis i e dg e e up pe E Pods eee POSE te ee ewe ees
121. egular pattern the same wells fail repeatedly Electrophoresis or pipetting error Defective capillaries arrays Autosampler is misaligned Refer to the instrument user guide for information on troubleshooting capillaries arrays Noise peaks are detected as size standard peaks Noise Contaminated sample The Peak Amplitude Threshold of the dye color associated with the size standard is set too high or low in the analysis method Prepare new sample Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 Adjust the analysis method so that the peak detection threshold associated is greater than the height of the noise signal See GeneMapper Software peak detection settings on page 94 Note You can adjust the threshold if you want to examine the data However we recommend that you rerun with increased size standard concentration Size call inaccurate for known DNA sample Incorrect size standard was added to sample Repeat with the correct size standard Size standard peaks are not sized correctly Examine the size standard peaks and compare the sizes to the size standard definition See Viewing the size standard definition on page 182 Migration issues See Migration troubleshooting on page 168 186 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting GeneMapp
122. en aes 109 Applications 26 TTT 110 Instrument and consumable recommendations cece eee eee eee 111 Experiment and primer design recommendations n n 00 cece cece eee e eens 111 Workflow m inaleehIe M eR ER bg tae patra TTT 112 jataatialysiS eroe s terete eed rope dope EQUI LENS pce dcn dere 112 Common problems with microsatellite analysis 00 ccc eect cee eeee 113 Identifying stutter products in microsatellite analysis cece cece e eee eee 113 OVERVIEW E Sek seth A AE EMM 113 Estimating the amount of stutter 2 2 2 2 0c eee sss 114 Dinucleotide repeats 5 ec RE od wa ee ed Se a gee EA eae 115 Evaluating data with stutter 2 22 2 6 eee eee eens 117 Is stutter a real problem 2 0000 cece eee eee eee e enna 118 For MOresINTOrMAtON e axe T Leste pete tine buie e eei irte Ant 118 CHAPTER 7 Single Nucleotide Polymorphism SNP Genotyping 119 Overview of SNP genotyping 0 20c cece rnrn eee teen n 119 lc 119 Applications SNP c eter be RUEDA e EUER HUE ages RAER D 119 SNaPshot Multiplex System sssssssssssssses e n n 120 COMPONENTS sooo desee bitu cec ub eet Tete erat eter t etat niat tal x 120 Principle of the analysis J L keeper tese DEE Ane Gael FAERIT RE 121 Advantages RM 121 Applications SNaPshot 5 ce Lebe eap be ears 121 Instrument and consumable recommendations cee eee eee tenet eens 122 Experiment and primer design recommendations c
123. en dyes by subtracting out the spectral overlap between the different dyes for more information see Multicomponent analysis with fluorescent dyes on page 36 9 10 11 12 13 Intensity vs Bin Number After spectral calibration IT Spectral calibration matrix standards are available from Life Technologies in premixed form for all Life Technologies instruments and dye sets see Table 18 on page 86 The values in a matrix generated by a spectral calibration are unique for each instrument for each dye set and for each specific set of run conditions The Data Collection Software applies the values in the matrix to the sample data to perform multicomponent analysis the separation of the dye fluorescence in the raw data from the instrument to the data stored in the sample files Note For 310 instruments you must manually create and apply the matrix file in the GeneMapper Software Refer to the instrument user guide for your instrument for instructions on performing a spectral calibration or creating a 310 matrix When to perform Perform a spectral calibration run When you use a new dye set on the instrument When you change the capillary array or polymer e For each combination of capillary array length and each dye set that you use After the laser or CCD camera has been realigned replaced by a service engineer f you observe a decrease in the quality of raw or analyzed data for example pull up and or pull down p
124. en the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure the dye labels do not affect PCR efficiency The procedure for creating custom dye sets is the same as the procedure for the 3730 Series instruments Creating a custom dye set on page 73 Refer to the specification sheet for your instrument to select a combination of capillary array and polymer that provide the required resolution see Instrument documentation on page 199 DNA Fragment Analysis by Capillary Electrophoresis 75 310 instruments Run modules and Note If you use GeneScan 600 LIZ Size Standard optimize the run module before performance use See Optimizing the 310 instrument run module on page 46 Table 15 310 instrument run module Fragment analysis run Resolution Genotypes day modules Samples day GS STR POP4 1 base detection up to 250 bases with 0 15 SD 4 dye gt 57 720 genotypest 2 base detection 250 to 350 bases with 0 3 SD 5 dye gt 57 960 genotypest 15 genotypes run 20 genotypes run Dye sets and Refer to the specification sheet for your instrument to select a combination of capillary array and polymer that provide the required resolution see Instrument documentation on page 199 Table 18 on page 86 lists the dye sets and matrix standards each instrument matrix standards Optimizing sample loading concentration 76
125. eneMapper Software includes a Microsatellite Default analysis method that you can use as a starting point for analysis Figure 22 on page 113 shows a typical microsatellite electropherogram from the GeneMapper Software The number of repeats for a given locus may vary resulting in alleles of differing lengths The following figure shows two different FAM dye labeled human dinucleotide loci from the GeneMapper Software tutorial dataset from two individuals The top panel illustrates a DNA sample that is homozygous at both loci a single major peak is observed at each locus the bottom panel shows a DNA sample that is heterozygous at the same loci two major peaks are observed at each locus DNA Fragment Analysis by Capillary Electrophoresis Chapter 6 Microsatellite Analysis S Figure 22 Example of microsatellite analysis of two samples by capillary electrophoresis Samples have different genotypes as shown by the different peaks for the same marker Genotypes Plot aax Eile Edit View Tools Alleles Help x Plot Setting Microsatelite Default w EE Panes 2 ijt uf N NS n LI TEE ILE Om Common problems with microsatellite analysis The most commonly encountered problems during microsatellite analysis are e Poor or non specific amplification See Optimizing PCR on page 55 and PCR troublesho
126. ent 3500 Series instrument 3730 Se ries instrument genetic diversity fingerprinting application 17 microsatellite application 16 genetic maps for new species fingerprinting application 17 genome scans 110 guidelines PCR contamination 64 PCR parameters 63 primer design 29 size calling 90 H hairpin secondary structures 31 HiCEP 136 applications 136 overview 136 principle 136 workflow 136 HID See Human Identification Hi Di formamide issues caused by improperly stored 159 storage conditions 82 high coverage expression profiling See HiCEP hot start PCR description 60 enzyme 23 thermal cycler parameters 60 62 Human Identification applications Go to lifetechnolo gies com humidity problems caused by 161 l injection factors affecting 78 time optimizing 79 voltage optimizing 80 instruments for use with guide 13 plate types 58 problems caused by 160 RFU ranges 158 run modules 46 signal intensity ranges 158 213 troubleshooting 158 159 180 Inter simple sequence repeat See ISSR ionic strength of buffer 78 ISSR applications 138 creating multiple bins 139 data analysis 139 example data 140 experiment and primer design recommendations 139 microsatellite application 16 overview 137 principle 137 workflow 139 L labeling fluorescent 25 laboratory water problems caused by contamination 159 linkage groups among crosses fingerprinting application 17 linkage mapping fragment analysis 16 micro
127. equency of a repeating unit can vary across a particular chromosome as shown in the following figure DNA Fragment Analysis by Capillary Electrophoresis 109 Figure 21 SSR density in exonic intronic and intergenic regions on individual human chromosomes lal monomers b dimers c trimers d tetramers lel pentamers f hexamers Blue bars exons red bars introns yellow bars intergenic regions http www ncbi nlm nih gov pmc articles PMC151303 figure F2 Copyright 2003 Subramanian et aL licensee BioMed Central Ltd This is an Open Access article verbatim copying and redistribution of this article are permitted in all media for any purpose provided this notice is preserved along with the article s original URL Applications 110 3 METE EEH 12345678 91011213141516171819202122 X Y 71234596789 1011111415161721819202122 X Y Chromosome Chromosome c d 2000 4 500 anoo Timers aooo Tare _ 1 000 NET Z 1 000 3 3000 B 1 200 D 2 400 1000 PT zo B w 150 400 20 o 123456 7 B 9101121141516171819202122 X Y Chromosome Pentamers 1234567 891011213141516171819202122 X Y Chromosome The large selection of highly informative markers has made microsatellite analysis a widely accepted tool for the following types of studies Linkage mapping studies Association studies Population studies Parentage analysis Breeding Custom microsatellite assays are often
128. er Software peak start and end settings 0 00 c cece cece nee ences 97 How the GeneMapper Software performs sizing ccc cece cece cece ee 98 Size standard dennin E aa R e Gar a SE eee nn 98 Step T SIze matching zo esu sank hae ODER ee eek IISQUE ree oe see 98 Step 2 Sizing curve and sizing 0 cece cece eee tee eetettteeteeee tees 100 Factors thataffect sizing 2x gee R 0 K nae eed EAE weds wee ae ed 100 GeneMapper Software sizing methods 0 c cc cece cece ee 100 least Squares method x iso notte st n ia a n ie eid Im uber cote teeth 100 Cubic Spline Interpolation method 000 cece ee 101 Local Southern method ac cere EDLID MERE EAT ed nU DER dis ee eis 102 Global Southern Method 5 eee see ese eed Shee Eda eee ed 103 Evaluating data gua 0 0 0 c cece ccc an 104 Examining PQVS 22 8 ate bird Wot ad oa ee en verbum ibit 104 Criteria for a good electropherogram cece cette teenies 104 Examining peak definitions ccc eee eens 105 Comparing data rese a wee RE R ead Seat dd e ERES RI 105 DNA Fragment Analysis by Capillary Electrophoresis 7 Contents S CHAPTER 6 Microsatellite Analysis 00 cece eee 107 Overview of microsatellite analysis n siiis 107 Principle ofthe analysis iene eke Rete tI Ett 108 Advantages of using microsatellite markers loci in genetic studies 108 Microsatellite motifs and distribution 0 e cece ete eee e
129. er Software troubleshooting Some problems with data can be caused by the settings used to analyze the data If peak height morphology and number of expected peaks are acceptable but the sample fails sizing it may be caused by analysis method settings that are not optimized for your application For additional troubleshooting information refer to the GeneMapper Software Help and the GeneMapper Software Reference and Troubleshooting Guide Pub no 4403673 Symptom Possible Cause Action GeneMapper Software error message 9 There are sample s that do not meet analysis requirements Please see Error Message in the Info view of each sample Do you want to continue Generic message the error View the Info tab see Examine the sample info raw data for each sample may be and EPT trace on page 154 different For information on resolving the error refer to the GeneMapper Software Reference and Troubleshooting Guide Error Message The bin set in the analysis method does not match the panel used for analysis The bin set in the analysis Modify the Bin Set selection on the Allele tab in the method does not match the analysis method panel used for analysis Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality piao r Marker Repeat Type S we f r B ds Haa 1 C Use marker specific stutter ratio if available 3 Mcros
130. er fields do not correspond to the dye colors displayed above the Calibration Peak Order fields n Example 1 on the next page if the order of the peaks in the calibration standard you are using is Orange Red Yellow Blue Green Purple specify for Calibration Peak Order 6 Orange 4 Red 3 Yellow 1 Blue 2 Green 5 Purple n Example 2 if the order of the peaks in the calibration standard you are using is Orange Red Yellow Blue Green specify for Calibration Peak Order 5 Orange 4 Red 3 Yellow 1 Blue 2 Green DNA Fragment Analysis by Capillary Electrophoresis 71 In Example 3 if the order of the peaks in the calibration standard you are using is Orange Red Green specify for Calibration Peak Order 3 Orange 2 Red 1 Green Expand the Parameters section then specify remaining settings Example 1 3 Arrange Dyes 1 2 4 5 6 Dye Selection Reduced Selection Calibration Peak Order Example 2 Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order Example 3 Arrange Dyes Dye Selection Reduced Selection vi v v v v iv Calibration Peak Order 0 zs sl 212 al E j 5 Perform a spectral calibration using the AnyDye dye set 6 Create an instrument protocol that specifies the custom dye set then specify the instrument protocol in an assay For more Refer to the specification sheet fo
131. erated with SNaPshot Multiplex Systems DNA Fragment Analysis by Capillary Electrophoresis Principle of the analysis Advantages Applications SNaPshot Chapter 7 Single Nucleotide Polymorphism SNP Genotyping Additionally the SNaPshot Primer Focus Kit allows rapid assessment of potential SNP oligonucleotides You can preview all potential single base extension products and calculate the mobility rate for each allele After assessing this data you can determine the optimal combination of SNP markers for multiplexing After you determine the multiplex format you can use the reference data created with the Primer Focus Kit to establish markers and bin sets in the GeneMapper Software and reduce the time required to define and edit bins manually In the single base extension technique a unlabeled primer is designed to anneal to the sequence adjacent to the SNP site After the primer anneals the single base extension occurs by the addition of the complementary dye labeled ddNTP dye terminator to the annealed primer Each of the four ddNTPs is fluorescently labeled with a different color dye Figure 29 Figure 29 Single base extension with dye labeled ddNTPs Dye labeled Unlabeled primer ddNTPs ee Ka K Kaea Ka a K a lKa K M H9 Template DNA The addition of ddNTPs yields marker fragments for the different SNP alleles that are all the same length but vary by color After electro
132. erform a water wash on all components of the system using the wizard in Data Collection Software 2 Replace reagents with Life Technologies products Broad lagging peaks Old or clogged capillary array Replace the capillary array or flush the capillary array with polymer Tailing peaks Degraded or improperly stored Hi Di Formamide Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 170 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible cause Action Uneven peak heights in dyes in multiplexed sample IL Ni Sample preparation issues Optimize sample preparation and PCR Preferential amplification of PCR products see PCR troubleshooting on page 176 Selection of dyes is not optimal for example a low intensity sample peak is labeled with a low intensity dye Select appropriate dye For information see Points to consider when selecting dyes for custom primers on page 39 Concentration of some samples is too high Adjust the pooling ratio before PCR see Multiplexing pooling strategies on page 27 e f overall concentration is too high dilute pooled samples with deionized water before PCR Increasing the MgCl concentration of some samples can reduce the disparity in peak heights but may also increase the amplification
133. esis 199 Peak Scanner Software documentation Document Publication number Peak Scanner Software Reference Guide 4382253 Peak Scanner Software Quick Reference Card 4383719 Application documentation Docament Publication number Reconstituting and Diluting Primers and TaqMan Probes 4370992 AFLP applications GeneMapper Software Getting Started Guide AFLP9 System Analysis 4403620 AFLP Microbial Fingerprinting Protocol 402977 AFLP Plant Mapping Protocol 4303146 Aneuploidy applications Aneuploidy Detection by QF PCR of STR Markers on the Applied Biosystems 3500xL Genetic Analyzer 106AP28 01 BAC applications BAC Fingerprinting on the Applied Biosystems 3730 3730xl DNA Analyzer 107AP04 01 Sizing of Large DNA Fragments Generated by BAC Fingerprinting on Capillary Electrophoresis System 106AP25 01 HiCEP applications High coverage gene expression profiling on the Applied Biosystems 3500xL Genetic Analyzer C015884 ISSR applications ISSR Genotyping of Endangered Plants Using an Optimized Workflow 106AP31 01 LOH applications GeneMapper Software Getting Started Guide Loss of Heterozygosity LOH Analysis 4403621 Relative Fluorescent Quantitation on CapillaryElectrophoresis Systems Screening for Loss of Heterozygosity 106AP15 01 in Tumor Samples on the Applied Biosystems 3130 Series Genetic Analyzers with GeneMapper Software v3 7 Methylation applications
134. ew current trace is blue GA ar re ga3100 Fun Hstory EPT Views Sesect run to vite Fe Customer Training 2006 04 25_15 01_O181 Current signal should be horizontal 20 Tene Elapsed minutes Air bubble in lower polymer Remove bubbles Refer to the instrument user guide for block information Clogged capillary caused by Flush the capillary array with polymer sample overloading Instrument has stopped Arcing to conductive surface Ensure that the ambient temperature is 15 to 30 C and the running and red light is on on the instrument humidity is 8096 Check for excessive condensation on the There are black marks instrument U Une SINE Arcing caused by bubbles in Remove bubbles from polymer polymer GeneMapper Software EPT view current trace is green Current signal shape should resemble the example shown in Examine the sample info raw data and EPT trace on page 154 DNA Fragment Analysis by Capillary Electrophoresis 181 Sizing or Size Quality SQ troubleshooting Viewing the size standard definition Modifying the size standard definition 182 Sizing issues can occur if the peaks detected do not match the peaks listed in the size standard definition for example if additional peaks are detected as size standard peaks or if size standard peaks are not detected To view the peaks detected in the size standard and the pea
135. factor calculated in 3500 Series Data Collection Software Use environment Multiuser client server deployment Remote auto analysis and command line operation Support Fully supported by Life Technologies DNA Fragment Analysis by Capillary Electrophoresis 91 Peak Scanner Software Features Overview Features 92 Peak Scanner Software is a nucleic acid sizing software that identifies peaks and fragment sizes for application specific capillary electrophoresis assays This software allows you to annotate data with functions such as labeling merging and splitting peaks The software stores all editing and analysis data in the original fsa data files generated on Life Technologies genetic analysis instruments Peak Scanner Software is available free of charge on www lifetechnologies com Note Life Technologies does not support Peak Scanner Software Use this software with data generated on 3730 Series 3130 Series and 310 instruments Itis not compatible with data generated on the 3500 Series instrument which performs fragment sizing during data collection Import and analyze fragment analysis sample files fsa from all currently supported Life Technologies genetic analyzers e Analyzed data sizing information is written back to the sample files fsa Ability to organize the sample files in a project e Simultaneous viewing of raw and analyzed data Large fragment sizing up to 1200 bp Abili
136. ffect PCR efficiency sample resolution and signal intensity Expired PCR reagents can cause decreased DNA template concentration Improperly stored Hi Di Formamide can cause Incomplete denaturation of both the size standard and sample peaks e Altered pH of the loading solution Tailing peaks e Artifacts e Decreased signal Ensure that you do not contaminate Hi Di Formamide when setting up samples For more information see Hi Di Formamide storage on page 82 Degraded or expired polymer or polymer that is left at ambient temperature for 77 days can cause Reduced capillary array life the number of runs per array Reduced resolution due to increased conductivity often caused by the hydrolysis of urea in the polymer Low current e Artifact peaks from degraded polymer Reduced sizing precision Sizing differences between various types of polymer are more apparent for sequences 50 bp Fragments 50 bp run on 3730 3730xl instruments with POP 7 polymer may have slightly lower sizing precision Polymer that is left at ambient temperature for extended periods of time can cause microbubbles in the pump Cold polymer can cause bubbles Ensure that polymer is at room temperature Allow polymer to equilibrate to room temperature and pressure Loosen the lid seal at least 30 to 60 minutes before use Do not leave the lid off the polymer bottle as dust may contaminate stock causing spikes in data
137. fication Module 402942 AFLP Amplification Core Mix Module 402005 SNaPshot Kits SNaPshot Multiplex Kit Contact your local representative SNaPshot Primer Focus Kit 4329538 GeneAmp EZ rTth RNA PCR Kit N808 0178 GeneAmp PCR Carryover Prevention Kit N808 0068 AmpErase UNG N808 0096 T For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Other user supplied materials Item Source Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Vortex MLS Millipore VS filter Millipore Part no VSWP 02500 CEPH Individual 1347 02 Control DNA 403062 T For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 198 DNA Fragment Analysis by Capillary Electrophoresis Documentation and Support The publication numbers in this section are for the latest product versions available at the time of publication For documentation for newer product versions g
138. g temperature is not known This method incrementally decreases the annealing temperature in early cycles to maximize the yield of specific products 2 Tmp x 20 2 Tmp x 10 94 0 94 0 0 15 N 65 07 0 15 0 30 55 0 E 0 30 Method Touchdown PCR XL PCR Use this profile for amplification of 5 to 40 kb PCR products using rTth DNA Polymerase XL and unique reaction conditions By providing longer templates XL PCR complements technologies for rapid long range PCR More complete genes can be amplified in one reaction from known expressed sequences allowing more introns to be spanned You can use XL PCR to amplify the control target a 20 8 kb product from Lambda DNA supplied in the kit 1 Hld 2 Tmp X 16 2 Tmp X 12 2 Holds 94 0 94 0 94 0 72 0 10 00 10 00 Method XL PCR Optimizing thermal cycling parameters Optimizing Six independent temperature blocks are available for the Veriti Thermal Cycler Each temperature block provides precise control over thermal cycling parameter optimization For information refer to our website To find the optimal thermal cycling parameters perform a series of runs varying the annealing or denaturation temperatures in 2 C increments Note Do not vary more than one parameter at a time Annealing temperature Positive effects Negative effects change Decreased Increased PCR product yield Increased amplification of non specific products background Increased Incr
139. gment lengths nt 17 fragments 47 93 292 695 51 99 317 946 55 126 439 82 136 557 85 262 692 t If run under denaturing conditions Figure 15 on page 52 fragments run 18 nucleotides shorter than the lengths listed above and some or all of the peaks appear split Do not use this fragment for sizing See Peaks not used for sizing on page 43 for information Both strands of the GeneScan 1000 ROX Size Standard fragments are labeled and are used for non denaturing applications DNA Fragment Analysis by Capillary Electrophoresis 51 Figure 15 GeneScan 1000 ROX Size Standard run under denaturing conditions Fragments run 18 nt shorter than lengths obtained under non denaturing conditions see the table on the previous page Under denaturing conditions sizing is not accurate above 539 nt therefore the and 674 nt corresponds to the 692 nt peak is not shown in the figure 118 539 244 X 274 o0 c o uw No SSS 1 AS LT Note Under denaturing conditions the two strands of this doubly labeled size standard migrate at different rates appearing as split peaks To ensure sizing precision and a reliable size standard definition you must define one peak from each split peak pair in the size standard definition J GM Ordering custom primers from Life Technologies You can obtain custom 5 end labeled primer
140. harge on www lifetechnologies com Use this software with data generated on 3730 Series 3130 Series and 310 instruments It is not compatible with data generated on the 3500 Series instrument which performs fragment sizing during data collection DNA Fragment Analysis by Capillary Electrophoresis 19 20 DNA Fragment Analysis by Capillary Electrophoresis Experimental Design Experimental design considerations 6 6 6 21 DNA polymerase enzyMe S 0 eee eee 22 Fluorescent labeling methods 6 6 c cece tenes 25 Singleplexing versus multiplexing e eee eee eee eee ee 26 Primer design guidelines 2 0 66 29 Dyes TTA fs al E tM LE L De 36 Dye sete noce rci ees be RC Gn RI RA a Fels a Miles a I e c edo a ea Ey 41 Size standards oe eeu bx eb ert putre ue 42 Ordering custom primers from Life Technologies 00000008 52 Testing the primers and optimizing conditions with test DNA panel 52 Experimental design considerations Consider the following questions when designing your experiment What sequences and markers loci are you investigating not applicable for AFLP studies Which enzyme is appropriate for your experiment see DNA polymerase enzymes on page 22 What is the expected allele distribution determine from published literature or from your own design and empirical testing What labeling method will you use see Fluorescent labeling methods on p
141. he DNA Although many protocols describe simple or fast extraction or purification methods carefully evaluate any changes or improvements in extraction or purification methods Also be sure that the physical and chemical condition of the sample itself are adequate for the intended labeling and assay methods IMPORTANT These items should never leave the PCR Setup Work Area e Calculator Gloves disposable Marker pen permanent Microcentrifuge e Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation e Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors Tube decapper autoclavable e Vortex DNA Fragment Analysis by Capillary Electrophoresis Amplified DNA work area Avoiding contamination from the environment Avoiding PCR product carryover Chapter 3 Optimizing PCR IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems s Veriti 96 Well Thermal Cycler s GeneAmp PCR System 9700 with the Silver 96 Well Block e GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block To avoid general contamination take the following precautionary measures Change pipet tips between samples Use filter plugged pipet tips Clean any work contaminated surface using a cloth soaked with 50 bleach IMPORTANT Before cleaning the sample block of a t
142. hen likely to be calculated incorrectly as well Note This method does not determine the amount of sizing accuracy error The Local Southern method determines the sizes of fragments by using the reciprocal relationship between fragment length and mobility as described by E M Southern 1979 IMPORTANT For the Local Southern Method to work you must have at least two size standard fragments smaller than your smallest unknown fragment and two size standard fragments larger than your largest unknown fragment If you do not a second order least squares curve extrapolation will be used to derive the size curve instead of the method specified in the analysis method Local Southern sizing curve Best Fit 2nd Order Curve AO 1 990206E 02 Al 2 075795E 01 A2 1 545595E 05 R 2 1 000 Size Calling Curve Local Southern Method T T T T T T T T T 1200 1400 1600 1800 2000 2200 2400 2600 2800 Local Southern Method equation L c m m0 LO The equation attempts to describe the reciprocal relationship between the mobility m and the length LO of the standard fragments How the Local Southern method works This method which is similar to the Cubic Spline method uses the four fragments closest in size to the unknown fragment to determine a best fit line value Only the region of the size standard near the fragment of unknown length is analyzed Note Size estimates may be inaccurate if any of the size stan
143. her you choose the 2nd or 3rd Order Least Squares Method in the Analysis Parameters dialog box the resulting size curve is either a quadratic or a cubic function The software uses the known standard fragments and the associated data points to produce a sizing curve based on Multiple Linear Regression DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software In the following figures you can see that in nearly all instances the mobility of an individual DNA fragment is coincident with the best curve fit of the entire data set Stated differently the mobility of most DNA fragments is strictly length dependent This method automatically compensates for fragments that run anomalously GeneMapper Software calculates a best fit least squares curve for all samples regardless of the sizing method you choose The curve is black in the Standard Sizing Curve window Note All of the graphs in this section were generated using GeneScan Software v3 7 1 These results are similar to results obtained when you use GeneMapper Software v3 5 and higher 2nd Order Least Squares sizing curve 4 R 2 1 000 Best Fit 2nd Order Curve AO 9 771623E 01 Al 1 220838E 01 A2 4 856885E 06 Size Calling Curve 2nd Order Least Squares 3rd Order Least Squares sizing curve Best Fit 3rd Order Curve AQ 1 266506E 02 Al 1 608974E 01 A2 2 041442E 05 A
144. hermal cycler refer to the instrument user guide for the proper procedure Close sample tubes when not using them e Always run a no DNA negative control A negative control contains no template DNA only primers and the DNA diluent usually water or buffer Aliquot reaction reagents to minimize the number of times you use a stock solution PCR product carryover is the contamination of an unamplified sample with previously amplified DNA Why carryover is a particular concern PCR product carryover is a particular concern because amplified PCR product is an ideal template for subsequent amplifications of that same target A single PCR amplification produces a large number of copies as many as 101 The inadvertent transfer of even a tiny volume or aerosol of amplified product can significantly contaminate unamplified samples Contamination can result in false positives and the detection and amplification of the contaminating sequence instead of the target sequence Precautionary measures Use positive displacement pipettes or filter plugged pipette tips Physically separate reactions before and after amplification Handle pre and post PCR solutions with separate sets of Pipettes Pipette tips Microcentrifuge tubes Gloves Use AmpErase UNG in reaction mixtures to prevent the subsequent reamplification of dU containing PCR products DNA Fragment Analysis by Capillary Electrophoresis 65 For more Life Tech
145. horesis 173 Symptom Possible cause Action Data spikes OE 438 FiterF TestMHA 007 56M Plus 05 152 SOM Plus E 436 FiterF TestMHA D07 5M Plus 029 153 SOM Plus LIUM 437 FiterF_TestHA_007_SGM_Plus_058 fsa SGM Plus Z Bubbles dried polymer or dust 1 Flush the water trap Refer to the instrument in the capillary array migrate user guide for information past the camera 2 Check for bubbles and run the bubble wizard if any are visible Clean all connections and tubing around the instrument pump 3 Check the polymer bottle the area around the pump lines and the array port for crystals If present warm the polymer gently to 30 C with gentle mixing then refill the pump and array with the polymer 4 f the problem persists perform a water wash and replace the polymer 174 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible cause Split peaks 165 165 170 UO T4 Withoutextensionstep A 5 5 3 RRR Ao 100 A O Ow 166 168 170 172 174 900 176 178 180 182 184 136 188 wo With extension step 700 T ml al A 300 200 mI ole 7 AR WAV A A Plus A and or minus A peaks due to Too many fragments for A addition e Suboptimal PCR conditions and or suboptimal
146. ic primer Conversely if binding is strongest at the 5 end the typical binding event on the template DNA begins at the 5 end Polymerases however cannot begin elongation until the 3 end binds Therefore the entire primer is used to distinguish among target sequences Also when performing a computer assisted search to evaluate binding to secondary sites in the target DNA consider the potential for gapped duplex formation A gapped duplex can form when the primer and target are completely complementary except for a single base Miller Kirchoff et al 1987 Miller Wlodawer et al 1987 Note Binding to secondary sites can also involve the formation of stable non Watson Crick base pairs Topal and Fresco 1976 Stable base pairing is most likely to occur between G and T but A C and G A pairs can also be stable Hunter 1986 All software programs have difficulty modeling these sorts of interactions Complementary sequences between two primers especially at the 3 ends can lead to the formation of product artifacts arising from amplified primer dimers and primer oligomers Avoid primers with inter complementary regions between members of a primer pair or pairs DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Post amplification Adding extensions that are not complementary to the template at the 5 end of the manipulations primer can facilitate a variety of useful post amplification mani
147. ies inhibit polymerase activity providing an automatic hot start while a thermostable accessory protein enhances specific primer template hybridization during every cycle of PCR This combination improves the fidelity of Jaq DNA Polymerase by two fold and is ideal for high throughput screening and multiplex PCR AccuPrime Tag DNA Polymerase broadens primer annealing temperatures giving you optimal performance between 55 C and 65 C Applications Multiplex PCR TOPO TA Cloning allele specific amplifications AccuPrime Taq DNA Polymerase High Fidelity Amplifies nucleic acid templates using antibody mediated hot start a blend of Taq DNA Polymerase and proofreading enzyme and AccuPrime accessory proteins for improved PCR fidelity yield and specificity over other hot start DNA polymerases This enzyme provides e The highest specificity and yield for the most robust PCR amplification e 9 fold higher fidelity than Tag DNA polymerase alone Minimal optimization steps even with non optimized primer sets Efficient amplification of targets over a broad size range up to 20 kb High fidelity is achieved by a combination of Platinum anti Tag DNA polymerase antibodies that inhibit polymerase activity providing an automatic hot start and the proofreading 3 5 exonuclease activity enzyme Pyrococcus species GB D The thermostable AccuPrime accessory proteins enhance specific primer template hybridization during every cycle of
148. ific amplification Poor quality or degraded DNA template Use fresh template Run an agarose gel to check sample concentration and quality If DNA is stored in water check water purity Insufficient or excess template DNA Use recommended amount of template DNA Run an agarose gel to check sample concentration and quality PCR inhibitors in the DNA sample binding proteins salts that carry over from poor DNA extractions Use different extraction procedures Incorrect thermal cycling parameters Check protocol for correct thermal cycling parameters Incorrect pH Use correct concentration of DNA and buffer Tubes loose in the thermal cycler Push reaction tubes firmly into contact with block before first cycle Third party or non PCR tube type used Use GeneAmp Thin Walled Reaction Tubes with Caps with Life Technologies thermal cyclers Primer concentration too low Use recommended primer concentration Primer design See Non specific amplification on page 32 Poor amplification non specific amplification in restriction ligation experiments AFLP only Incomplete restriction ligation in experiments involving restriction ligation due to insufficient or insufficiently active ligase 1 Test the ligase activity with control DNA 2 Repeat restriction ligation with a higher concentration of ligase in Weiss units Note 1 Weiss unit 67 c
149. ignal 167 Size standard signal and sample signal are not balanced 168 Migration issues TRE S Size standard peaks are not migrating as expected during 168 a normal run time Sizing precision is low 168 Abnormal peak morphology Symptom See page Poor resolution 169 Loss of resolution 169 Broad lagging peaks 170 Tailing peaks 170 Uneven peak heights in dyes in multiplexed sample 171 Sudden loss of signal in all samples 171 Multiple dye colors are detected as one dye color 171 Extra peaks Symptom See page Extra peaks 172 Pull up peaks 173 Data spikes 174 Split peaks 175 Many small extraneous peaks appearing next to a high 175 intensity peak PCR Symptom See page Poor priming resulting in weak signal 176 Amplified DNA concentration is lower than expected 176 PCR inhibition 176 Contamination with exogenous DNA 176 Poor amplification nonspecific amplification 177 162 DNA Fragment Analysis by Capillary Electrophoresis Irregular baseline Instrumentation Sizing or size quality GeneMapper Software Chapter 11 Troubleshooting Symptom See page Poor amplification nonspecific amplification in restriction 177 ligation experiments AFLP only Hairpin secondary structures in PCR primers 177 Primer dimer formation 178 Symptom See page Constant elevated signal in raw data 178 Baseline waterfall 178 Noisy baseline 179 Adequat
150. ility Loss of Heterozygosity LOH Inter simple sequence repeat ISSR Multilocus Variant Analysis MLVA SNP Genotyping see Chapter 7 Single Nucleotide Polymorphism SNP Genotyping A Single Nucleotide Polymorphism SNP marker consists of a single base pair that varies in the known DNA sequence thereby creating up to four alleles or variations of the marker Applications include SNaPshot Multiplex Kit 16 DNA Fragment Analysis by Capillary Electrophoresis Chapter 1 Introduction to Fragment Analysis Fingerprinting see Chapter 8 Fingerprinting Several AFLP based technologies use restriction enzyme length polymorphism and polymerase chain reaction PCR to generate a fingerprint for a given sample allowing differentiation between samples of genomic DNA based on the fingerprint Applications include Microbial genome typing Animalor plant genome typing Creation of genetic maps of new species Genetic diversity and molecular phylogeny studies Establishment of linkage groups among crosses Relative Fluorescence see Chapter 9 Relative Fluorescence Quantitation RFQ Relative fluorescence applications compare peak height or area between two samples Common techniques include Qualitative Fluorescence QF PCR Quantitative Multiplex PCR of Short Fluorescent Fragments OMPSF Multiplex Ligation dependent Probe Amplification MLPA Applications include LOH in tumor samples
151. illary Array 22 cm 4 Capillary 3130 Genetic Analyzers 4333463 Capillary Array 80 cm 16 Capillary 3130xl Genetic Analyzers 4319899 Capillary Array 50 cm 16 Capillary 3130xl Genetic Analyzers 4315930 Capillary Array 36 cm 16 Capillary 3130xl Genetic Analyzers 4315931 Capillary Array 22cm 16 Capillary 3130xl Genetic Analyzers 4319898 GeneScan Size Standards See GeneScan size standards on page 197 Matrix Standard Kits See Matrix standards for spectral calibration on page 197 POP 4 Polymer 7000 uL 3130 3300xl Genetic Analyzers 4352755 POP 4 Polymer 3500 uL 3130 3300xl Genetic Analyzers 4363752 POP 6 Polymer 7000 uL 3130 3300xl Genetic Analyzers 4352757 POP 6 Polymer 3500 uL 3130 3300xl Genetic Analyzers 4363783 POP 7 Polymer 7000 uL 3130 3300xl Genetic Analyzers 4352759 POP 7 Polymer 3500 uL 3130 3300xl Genetic Analyzers 4363785 Running Buffer 10X 402824 MicroAmp Optical 96 Well Reaction Plate N8010560 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 Hi Di Formamide 4311320 310 Analyzer materials DNA Fragment Analysis by Capillary Electrophoresis 195 Itemt Source Capillary Array 47 cm 310 DNA Analyzer capillary array 402839 Capillary Array 61 cm 310 DNA Analyzer capillary array 402840 96 well tray adaptor for 9700 thermal cycler trays 4305051 GeneScan Size Standards See GeneScan
152. immis K N 2000 An evaluation of terminal restriction fragment length polymorphism T RFLP analysis for the study of microbial community structure and dynamics Environmental Microbiology 2 1 39 50 Naimuddin M and Nishigaki K 2004 Genome analysis technologies Towards species identification by genotype Briefings in Functional Genomics and Proteomics 1 4 356 371 Ronquist F and Huelsenbeck J P 2003 MRBAYES 3 Bayesian phylogenetic inference under mixed models Bioinformatics 19 1572 1574 DNA Fragment Analysis by Capillary Electrophoresis References Sakamoto M Takeuchi Y Umeda M Ishikawa I and Benno Y 2003 Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis J Med Microbiol 52 79 89 Savelkoul P H M Aarts H J M de Haas J et al 1999 Amplified fragment length polymorphism analysis the state of an art J Clin Microbiol 37 10 3083 3091 Serra R Cabanes F J Perrone G Venancio A Mule G and Kozakiewicz Z 2006 Aspergillus ibericus a new species of section Nigri isolated from grapes Mycologia 98 2 295 306 Shihabi Z K and Hinsdale M E 1995 Some variables affecting reproducibility in capillary electrophoresis Electrophoresis 16 2159 2163 Shinde D Lai Y Sun F and Arnheim N 2003 Taq DNA polymerase slippage mutation rates measured by PCR and quasi likelihood analysis GA GT n
153. ines on page 29 Artifact peaks See Pull up peaks from a sample appear in the red or orange dye signal and are detected as size standard peaks due to over saturation of sample peak signal on page 185 Sample or reagent contamination Use fresh sample or reagent Contamination with exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Renaturation of denatured samples Load the sample immediately following denaturation or store it on ice until ready 172 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible cause Action Pull up peaks T Iu SHS Maie aii Cnt Phi in A o f Incorrect or poor quality matrix or spectral calibration Run a new spectral calibration Edit the instrument protocol to specify the correct spectral calibration Wrong matrix or spectral use for analysis of 310 instrument data Reanalyze with the correct matrix in the GeneMapper Software Offscale saturated signal in primary peak caused by high sample concentration Decrease sample concentration Decrease injection time Polymer on instrument 7 days degraded polymer contaminants Perform warm water washles and replace polymer If the problem persists replace the capillary array DNA Fragment Analysis by Capillary Electrop
154. ing on page 98 Correct for injection to injection variations that result in differences when comparing the same DNA fragments from different capillaries runs and instruments When comparing fragment size across injections ensure that data is analyzed with the same sizing method and the same size standard definition Size standard peak For optimum performance the signal intensity of the size standard peaks should be intensity lower than or equal to the signal intensity of the sample peaks For more information see Balancing size standard and sample peak intensities on page 77 42 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Selecting a Select a size standard with at least two fragments smaller and larger than your GeneScan size unknown sample fragments and with a dye that is compatible with the dyes used for standard labeling primers LIZ Size Standard 5 dye chemistry ROX Size Standard 4 dye chemistry Expected marker GS120 GS500 GS600 GS1200 GS350 GS400HD GS500 GS1000 length Lizet Lizet LIZ t LIZ t ROX ROX ROX ROX page 47 page 50 page 45 page 47 page 49 page 49 page50 page 51 lt 120 bp z z V S lt 400 bp lt 500 bp E v 7 600 bp E E m 2 lt 1000 bp z z 1200 bp Used with SNaPshot Multiplex Kit T For
155. ing during multiplex PCR using high quality primers is particularly important For example the decreased specificity and thus the increased reagent consumption of one pair of degraded PCR primers can affect the entire multiplex reaction Although you can compensate for a degraded pair of primers to some extent by increasing the concentration of the other primer pairs the increased cost per reaction and the decreased reproducibility over time do not justify this short term solution When buying or making primers make sure that they are length purified and that they are free of contaminants DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Primer pair concentrations Typically start out with equal concentrations for all primer pairs It is often necessary to adjust the concentration of primer pairs in the multiplex reaction until the peak heights are relatively even Increase the primer pair concentration for fragments showing weak amplification Decrease the primer pair concentration for fragments showing significantly greater than average amplification Primer pair compatibility With either single or multiplex PCR evaluate primers for compatibility Avoid excessive regions of complementarity among the primers Also select or design primers with similar melting temperatures Tm After identifying compatible primer pairs test and evaluate pairs in singleplex reactions before attempting any multiplex
156. ing temperature of primer template binding than do A T bonds To ensure stable annealing of primer and template while avoiding problems with the internal secondary structure of primers or long stretches of any one base select primers with a 4076 to 6076 G C content Note Designing primers based on Tn and primer length to avoid primer dimers and SWS p m P 8 P gapped duplex structures is more important than designing primers based on actual percent G C content Effects of base order Two primer template complexes with identical content may have different Tn values because base order influences the overall annealing stability You can determine the exact effect of base order on complex stability using the base pairing energies listed in Table 4 adapted from Salser 1978 Larger negative values represent more stable interactions between the template and primer Table 4 Base pairing energies kcal dinucleotide pair 5 Nucleotide 3 Nucleotide A C G T A 1 2 2 1 2 1 1 8 C 2 1 4 8 3 0 2 1 G 2 1 4 3 4 8 2 1 T 1 8 2 1 2 1 1 2 Example Consider the two sequences 3 GAC 5 and 3 CGA 5 The sequence 3 GAC 5 contained within a primer would contribute 4 2 kcal to the binding energy 2 1 kcal 3 GA 5 2 1 kcal 3 AC 5 4 2 kcal However if the G and C are next to each other as in 3 CGA 5 the contribution increases to 6 4 kcal 74 3 kcal 3 CG 5 2 1 kcal 3 GA 5
157. internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing alifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners AmpErase AmpliTaq AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc AFLP is a registered trademark of Keygene N V DNeasy is a registered trademark of Qiagen 2012 Life Technologies Corporation All rights reserved Contents About This Guide 5 520 rN testes AN TAT NERNET E R RRR 12 REVISION RISTA TTT 13 Purpose ie neta atte 1 etii E ene ld fees atte D 13 Prerequisites cet des ear EZ E Rr e en Rie ool A tiU mies 13 Structure of this Guid e inre oat Ep eR DEM UD me baies te dte e euh yew ak 14 S CHAPTER _ Introduction to Fragment Analysis 15 Fragment analysis versus sequencing what is the difference 0 00 eee eee ee eee 15 I ragmientanalysiSa 3 on trucco Anke th can tates AE ak ited rechte 15 Sequen ngea etr nee TT 16 What can do with fragment anaves ee 16 Types of applications cssc Re Fed b n E ede A ees Tub EROS US 16
158. ion for the selected sample s Size Match Editor t3 File Edit View Tools xag 2011 05 11 10 05 1 Size Matches Size Calling Curve Sizing Qualty 1 0 240 340 280 399 260 160 so 140 200 20 320 19504 31 400414 440 1 420 460 15504 250 380 480 520 i B 580 11504 600 T sod 7504 3504 i L l L j T T T T T T T 1870 2270 2670 3070 3470 3870 4270 4670 5070 5470 5870 OK Cancel Apply 4 In the Navigation Pane of the Size Match Editor select a sample file to display the sizing data for the associated sample DNA Fragment Analysis by Capillary Electrophoresis 153 5 Review the data for the following qualities Signal strength The signal strength peak height of all peaks must exceed the Peak Amplitude Thresholds defined in the analysis method used to analyze the data e Correct size calls labels All peaks listed in the size standard definition must be correctly identified by the software The labels above the peaks must be in sequential order from left to right low to high e Evenness of signal strength All peaks should have relatively uniform signal strengths Note To magnify the plot of the Size Matches tab drag the mouse cursor Q across a region of the x or y axis Examining the raw Data for SO samples is displayed only in the Raw Data tab see Examine the data for 9 red SQ sample info raw data
159. ist of Calibrations for Dye Set drop down list select the spectral calibration you want to use The spectral profile and raw data is displayed a i l aaas S BE Dye Set Z BigDyeV 3 Active Calibration for LL Matrix used for Capillary 10 9 Wed Jun 04 14 28 16 Condition 3 652025 Active Calibration for Dye Set Z BigDye V3 Spectral_Z_withOverride List of Calibrations for Dye Set Z BigDyev3 Spectral z withoverride m ied Jun 04 14 05 23 PDT 2003 Wed Jun 04 14 28 16 PDT 2003 bap Spectral_Z_withOverride d Click Set e Create an instrument protocol that specifies the custom dye set For more Refer to the specification sheet for your instrument to select a combination of capillary information array and polymer that provide the required resolution see Instrument documentation on page 199 74 DNA Fragment Analysis by Capillary Electrophoresis 3130 Series instruments Chapter 4 Optimizing Capillary Electrophoresis Run modules and Note If you use GeneScan 1200 LIZ Size Standard download a new run module performance and optimize it before use See Downloading 3130 instrument run modules from our website on page 48 Table 14 3130 Series instrument run modules and resolution 24 hr throughputt Fragment analysis run Array Polymer Run 3130 3130xl Resolution Performance modules length time Analyzer Analyzer Spt GTt GTt F
160. ject Analyze the data by clicking the green arrow to analyze the samples in the project Review the data 1 Optional In the Genotypes tab review the Process Quality Value PQV columns BD BIN CC LPH OBA OS PHR SHP SP SPA SPU and XTLK 2 Review the size quality and sizing data In the Samples tab examine the Size Quality SQ scores and the size standards 3 Modify sizing or analysis parameters if necessary 4 Display the samples and genotypes plots 5 Optional Save print export the results 1 View sizes in sizing table and label and or edit peaks 2 Save project and print or export results if necessary 3 Check sizing quality 4 Optional Modify sizing or analysis parameters if necessary For a list of the GeneMapper Software documents available see Documentation and Support on page 199 DNA Fragment Analysis by Capillary Electrophoresis 93 GeneMapper Software peak detection settings Peak Amplitude Thresholds Smoothing Baseline Window Min Peak Half Width Polynomial Degree and Peak Window Size parameters 94 Only peaks with heights that exceed the peak amplitude threshold values for a dye color are detected Smoothing optimizes peak size and can reduce the number of false peaks detected None default applies no smoothing None is useful if the data display sharp narrow peaks of interest Light provides the best results for typical data
161. ks listed in the size standard definition for a sample select the sample then click Analysis gt Size Match Editor to view the sizing information for the selected sample s Size Match Editor File Edit View Tools X f 2011 05 11 10 05 1 Size Matches Size Calling Curve Sizing Quality 1 0 Override SQ 240 1 340 o 200214 16 l 80 140 360 20 320 19504 314 400414 440 1 420 460 45504 250 nu 480 520 sod E ENAN LuL 1870 2270 2670 3070 3470 3870 4270 4670 5070 5470 5870 i Ok Jl Cancel JI Apply J If the expected peaks are not detected your first course of action should be to determine the cause of the peak detection issue and resolve the issue If you want to use the data even if the size standard data is of lower quality you can modify the size standard definition described below to eliminate or add peaks to improve the size standard quality result Note Data for SQ samples is viewable only in the Raw Data view For more information on techniques for improving sizing accuracy on Life Technologies genetic analyzers refer to Rosenblum et al 1997 and Wenz et al 1998 1 In the Project Window select Tools K GeneMapper Manager 2 Select the Size Standard tab then select the size standard definition used to analyze the data 3 Select Save As and name the new size standard definition DNA Fragme
162. labeled The unlabeled strand does not interfere with peak detection of the labeled strand DNA Fragment Analysis by Capillary Electrophoresis 49 Figure 13 GeneScan 400HD Size Standard run under denaturing conditions LLL ALL 1 LLL JL UUL JLUL JL JL GeneScan Range 35 to 500 bp under denaturing conditions 900 ROX Size This size standard is recommended for analysis of tri and tetranucleotide Standard microsatellite loci which can often exceed 400 bp in length GeneScan 500 ROX denatured fragment lengths nt 16 fragments 35 139 250 400 50 150 300 450 75 160 340 490 100 200 350 500 Do not use this fragment for sizing See Peaks not used for sizing on page 43 for information Only one strand of the double stranded DNA fragments in this size standard is labeled The unlabeled strand does not interfere with peak detection of the labeled strand when run under denaturing conditions 50 DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Figure 14 GeneScan 500 ROX Size Standard run under denaturing conditions 440 T ant 160 200 x so 1397 2901 100 GeneScan 1000 Range ROX Size e 100 to 900 bp under non denaturing conditions Standard e 100 to 539 bp under denaturing conditions verified with POP 49 polymer only GeneScan 1000 ROX non denatured fra
163. lary Electrophoresis Contents Instrument and ambient condition issues 00 cece eee tenet eens 160 Gapillanyearray sai uri Ste tre co ee aie Rete tes tae hee sh cele sat AL acne a 160 Pump large bubbles eec rece ROREM R TR 160 Pump small bubbles ch ee ee ie ei ae RR eee wr lI RITE 161 Pump polymer leaks usse Lp b e EE RICE EGER DIRE EU deere PER 161 Autosampler misalignment cece cece eee cece ss ee 161 Temperature h midity a tec cnt etn ahaa en eee ATR aes 161 Matrix spectral IssueS 2 0 2 cece ene e e 161 Symptoms you may observe 000 cece cece n 162 Irregular signal intensity cepe N Ee AR std KaR KRL Meee edhe ge eee RISE 162 Migration iSSUCS 0c cece e eee eee e enna 162 Abnormal peak morphology 000 00 eee cece cece eee e eee eee ee eeeees 162 Extra peaks oz feb ae Ane cr fo ae Ge crn t erit o nee lel e Pa the oe Ale SAD 162 PGR ui een Pai Bae ENDO ORI IE ie te i es eiu 162 Irr gularibaseline su cig cbe E eat Rn LEVE p at Eee wees 163 Instrumentation eese eee errem Sie dee vd Bed ened Eo lm ie el da 163 Sizirig ot size quality iene yee ee recs ub els melius el t edet dh ats 163 GeneMapper Software 0 00 ccc ccc cence net e ent ent e ene en eens 163 Irregular signal intensity troubleshooting cece eee eee eee eens 164 Migration troubleshooting e ie cee eek ees eek eR ees be AREY eee S PNEU 168 Abnormal peak morphology troubleshooting 00 cece cece cece cece e eae 169
164. latinum antibody technology provides a simple automatic hot start method that improves PCR specificity PCR Enhancer Solution is included for higher primer specificity broader magnesium concentration broader annealing temperature and improved thermostablilty of Platinum9 Pfx DNA Polymerase The PCR Enhancer Solution also helps optimize PCR of problematic and or GC rich templates Platinum Pfx provides e 26 times higher fidelity than Tag DNA polymerase Amplification of fragments up to 12 kb Room temperature reaction assembly Applications Amplification of DNA from complex genomic viral and plasmid templates and RT PCR Unit Definition One unit incorporates 10 nmoles of deoxyribonucleotide into acid precipitable material in 30 minutes at 74 C SuperScript III Proprietary mutant of SuperScript Il RT that is active at 50 C and has a half life of 220 minutes Reverse providing increased specificity with Gene Specific Primers GSPs and the highest cDNA yield of Transcriptase RT all RTs It is ideal for RT PCR of a specific gene or generating cDNA from total or poly LAC RNA sample Like SuperScript II it synthesizes a complementary DNA strand from single stranded RNA DNA or an RNA DNA hybrid SuperScript III RT is genetically engineered by the introduction of point mutations that increase half life reduce RNase activity and increase thermal stability Applications array labeling cDNA libraries RT PCR primer extension
165. le cause Possible cause if the control fails Action Incorrect PCR thermal cycling conditions Choose correct temperature control parameters refer to your instrument user guide Pipetting errors Calibrate pipettes attach tips firmly and check technique Combined reagents not spun to bottom of Place all reagents in bottom of tube Spin tube briefly after combining DNA Fragment Analysis by Capillary Electrophoresis 157 Sample issues Sample concentration Sample contamination Salt concentration 158 Possible cause if the control fails Action Put tubes in block immediately after reagents are combined Combined reagents left at room temperature or on ice for extended periods of time Restriction digest incomplete AFLP applications Repeat the restriction ligation Make sure that the volume of enzyme added does not cause the amount of glycerol to be gt 5 which can lead to EcoRI star activity If the sample concentration is too low the signal to noise ratio may be too low to discriminate between sample peaks and background fluctuations If the sample DNA concentration is too high signal intensity may be off scale or saturated and can cause Split peaks Raised baseline Pull up peaks which can affect sizing and accuracy of genotypes Adjust sample concentration to ensure signal intensity is within the recommended range for your instrument Instru
166. lity to generate fragment sizes that are close to the actual size as determined by sequencing The size of a DNA fragment is altered by the dye with which it is labeled and each Life Technologies dye has a different size Therefore a fragment with a known size may be sized differently when run using Life Technologies dyes and instruments Although this size may not be accurate when compared to the actual size it will be precise when compared to other fragments run under the same conditions Note the following Sizing differences between various types of polymer are more apparent for sequences 50 base pairs bp e Smaller fragments 50 bp run on POP 7 polymer on 3730 3730xl instruments may have slightly lower sizing precision The size of a fragment is calculated based on the size standard with which it co migrates Dye labeled DNA fragments can yield different sizes when run with a different instrument polymer capillary array length or size standard as shown below High precision is important in relative sizing Sample 1 wn 310 instrument Aho S 200 a a AAA 100 Sample 1 3130 instrument a 200 122 44 e Use the same sizing method for all injections To verify check the analysis method in the GeneMapper Manager or in the Size Match Editor window e Use the same size standard for all samples in a run You may need to modify the size standard definition of individual samples To verify overlay th
167. low Modify the size standard definition and remove the size standard peak that overlaps with the primer peak Change the analysis range in the Advanced Peak Detection Algorithm field in the Peak Detector tab of the analysis method Select Partial Range and select a starting data point that eliminates the primer peaks from analysis primer read region J rpi LI Size standard peaks labeled in a ma 20 ga UTi Size standard definition modified to eliminate peaks labeled in primer read region anseo 40 ae LIlLELLETL ELIT EEEN fiin Larger size standard peaks are Run time too short Increase run time not present in trace Late start caused by a reagent issue a blocked capillary or high sample concentration Use fresh polymer A non Life Technologies size standard was used Use a Life Technologies size standard 184 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom SQ A or and Possible Cause Action Extra peaks in size standard trace Size Matches Size Calling Curve Sizing Quality 1 0 5200 4800 4400 4000 3600 3200 2800 2400 2000 1600 1200 800 400 3000 1 139 150 m 200 Pull up peaks from a sample appear in the red o
168. lso includes minisatellites Highly repetitive simple sequence repeats ranging from approximately 10 to 30 base pairs Minisatellites fall into the broader category referred to as variable number of tandem repeats VNTRs which also includes microsatellites In GeneMapper Software a collection of expected size ranges and dye colors markers associated with each primer pair Studies to determine evolutionary relationships between organisms for example species or populations An enzyme that catalyzes polymerization DNA and RNA polymerases build single stranded DNA or RNA respectively from free nucleotides using another single stranded DNA or RNA as the template A locus with more than one allele that is commonly found in a population DNA Fragment Analysis by Capillary Electrophoresis population study primer pull up peaks quantum yield restriction enzymes template Variable Number Tandem Repeat VNTR Glossary A study that types genetic markers on a large population to identify associations between a marker and a phenotype A short single strand of DNA that serves as the priming site for DNA polymerase ina PCR reaction Artifact peaks in one or more dye colors that are caused by high or saturated signal intensity in another dye color The probability that a dye in its excited state will emit a photon as it decays back to the ground state Endonucleases that cleave the phosphate backbone of double st
169. ly labeled fragments are separated and sized Size bp Intensity RFU DNA Fragment Analysis by Capillary Electrophoresis 15 Sequencing Sequencing is the determination of the base pair sequence of a DNA fragment by the formation of extension products of various lengths amplified through PCR For more information refer to the DNA Sequencing by Capillary Electrophoresis Chemistry Guide Pub no 4305080 Figure 2 Sequencing fluorescently labeled nucleotides are separated and base called Base assignment bp w 1408 T eto iee 144 iao iNo in yMO 170 1i 10 iero e i it T Tt T x T 6G T 6 T T T Ta 7 2 T Ea 8 id 14 14 T Intensity RFU lj M i 1 V M 106 Y U YU M N VY Y KR wi RAS mPANBERREEEPEENRENEBRRBNNRNRRERSMI What can do with fragment analysis Types of Microsatellite STR analysis see Chapter 6 Microsatellite Analysis applications Microsatellite markers loci also known as short tandem repeats STRs are polymorphic DNA loci consisting of a repeated nucleotide sequence In a typical microsatellite analysis microsatellite loci are amplified by PCR using fluorescently labeled forward primers and unlabeled reverse primers The PCR amplicons are separated by size using electrophoresis Applications include Linkage mapping Animal breeding Human animal and plant typing Pathogen sub typing Genetic diversity Microsatellite instab
170. lymer may have slightly lower sizing precision than expected 168 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Abnormal peak morphology troubleshooting Symptom Possible cause Action Poor resolution High sample concentration Dilute the sample before adding to formamide size standard mix Injection time and or voltage too high See Optimizing electrokinetic injection parameters on page 78 Wrong capillary array and or polymer used Use appropriate capillary array or polymer for your application Incomplete strand separation due to insufficient heat denaturation Make sure the samples are heated at 95 C for 3 to 5 minutes then immediately placed on ice for 2to 3 minutes before loading Loss of resolution Pump polymer block or septa contaminated with chemicals during cleaning 1 Perform a water wash Refer to the instrument user guide for information 2 Replace polymer buffer septa and water waste with fresh materials Incomplete replacement of polymer between runs 1 Check the polymer delivery system for leaks looking for residue in and around the polymer block area 2 Check the pin valve for signs of arcing on the tip Black markings within the block channel are also a sign of an arcing event 3 Check for polymer in the anode buffer jar If you see evidence of a leak retighten connections then run the sam
171. lymorphic DNA loci that contain a repeated nucleotide sequence Each repeat unit can be 2 to 7 nucleotides in length and alleles differ by the number of repeats The number of nucleotides per repeat unit is the same for a majority of repeats within a microsatellite locus Locus X CA HQ b CA C A CA La Allele 1 ual CA CAIG A C A IC A ag Allele 2 LC Microsatellite markers are also known as Short tandem repeats STRs e Simple sequence repeats SSRs e Variable number tandem repeats VNTRs DNA Fragment Analysis by Capillary Electrophoresis 107 Principle of the analysis Advantages of using microsatellite markers loci in genetic studies 108 Microsatellite analysis is the separation of fluorescently labeled fragments using forward and reverse primers and determination of the relative size of the fragments A PCR primer pair consists of two oligonucleotides forward and reverse primers typically 15 to 30 nucleotides long Each primer hybridizes to its respective complementary strand of the DNA template such that the primer pair flanks the region of interest Based on the application one or both of the primers may be labeled with a fluorescent dye The number of repeat units at a microsatellite locus may differ so alleles of many different lengths are possible at each locus The microsatellite marker in the figure below contains a dinucleotide repeat When PCR is performed using
172. lysis with GeneMapper Software and Peak Scanner Software on page 89 If noise peaks are detected increase the Minimum Peak Half Width or use a stronger smoothing option when analyzing noisy data You can determine the relative quantities of two 5 end labeled fragments by comparing the corresponding peak areas or peak heights on a GeneMapper Software or Peak Scanner Software electropherogram DNA Fragment Analysis by Capillary Electrophoresis 145 Assess the reproducibility of peak height and area for each new analysis Note the following Use area for slow migrating or wide peaks at high concentration e Use height for sharp peaks at low concentration There is a linear relationship between the migration time and the reproducibility As the migration time increases the peak width and area increase Therefore fast migrating peaks result in higher reproducibility as measured by the peak area However improved reproducibility calculated using peak height has been observed as the migration time increases Shihabi and Hinsdale 1995 Determini ng To determine the relative number of molecules of two different sized fragments relative number of calculate the ratio of respective peak areas or heights Make sure to compare peak area molecules to peak area or peak height to peak height Iftwo fragments are similar in size compare peak heights especially if the peaks overlap slightly If the peaks are well defined peak area and pe
173. m DNA Fragment Analysis by Capillary Electrophoresis 81 Optimizing run temperature Perform non denaturing applications at lower temperatures 27 to 42 C Protocols for denaturing applications use POP 4 or POP 7 polymer with optimized run temperatures Altering the run temperature can affect migration rates and resolution Other factors that affect electrophoresis Laboratory temperature and humidity Salt concentration ionic strength and conductivity Hi Di Formamide storage 82 Maintain the laboratory temperature between 15 to 30 C After the instrument is set up and in operation the laboratory temperature should not fluctuate more than 2 C The instrument can tolerate up to 80 non condensing relative humidity Avoid placing it near heaters or cooling ducts Salt anions compete with negatively charged DNA for entry into the capillary during electrokinetic injection As the salt concentration of a sample increases less DNA will enter the capillary decreasing the fluorescence signal Excess salt can also precipitate the DNA in the sample tube in the presence of formamide The amount of DNA injected is inversely proportional to the ionic strength of the solution Butler et al Et nr Hep ia Heo DNA ampiel Aputter sample DNA DNA __ is the amount of sample injected DNA ampie is the concentration of DNA in the sample inj E is the electric field applied Eh
174. ment Recommended sigga Fluorescence saturation level 3500 Series 175 10 000 RFU 30 000 RFU 3730 Series 150 10 000 RFU 30 000 RFU 3130 Series 150 4000 RFU 8000 RFU 310 150 4000 RFU 8000 RFU If necessary dilute PCR products before including the size standard in the reagent mix so that the final allele peak height falls into the recommended range for the instrument Sample contamination can mimic a degraded capillary You can determine if the capillary issue is caused by sample contamination by running a size standard and formamide only see Running controls to isolate a problem on page 156 Salt anions compete with negatively charged DNA for entry into the capillary during electrokinetic injection As the salt concentration of a sample increases less DNA will enter the capillary decreasing the fluorescence signal Excess salt can also precipitate the DNA in the sample tube in the presence of formamide DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Reagent and consumable issues Laboratory water PCR reagents Hi Di formamide Polymer IMPORTANT For every chemical read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Poor quality laboratory water systems and cleaning reagents can adversely a
175. mers who plan conduct and troubleshoot fragment analysis applications This guide is for use by novice and experienced users who perform automated fragment analysis with any of these instruments Applied Biosystems 3500 or 3500xL Genetic Analyzers 3500 Series instruments e Applied Biosystems 3730 or 3730x1 DNA Analyzers 3730 Series instruments e Applied Biosystems 3130 or 3130xl Genetic Analyzers 3130 Series instruments 310 Genetic Analyzers 310 instruments Prerequisites This guide assumes that Life Technologies genetic analyzers and other instruments for which Life Technologies provides installation service have been installed by a Life Technologies technical representative Life Technologies reagents are used DNA Fragment Analysis by Capillary Electrophoresis 13 Structure of this guide Chapter Subject Introduction 1 Introduction to Fragment Analysis 2 Experimental Design Core processes in fragment analysis 3 Optimizing PCR 4 Optimizing Capillary Electrophoresis 5 Data Analysis with GeneMapper Software and Peak Scanner Software Types of fragment analysis 6 Microsatellite Analysis 7 Single Nucleotide Polymorphism SNP Genotyping 8 Fingerprinting 9 Relative Fluorescence Quantitation RFQ 10 Additional Applications Troubleshooting 11 Troubleshooting Reference information Ordering Information Documentation an
176. mor bottom panel samples the peak height of the larger 124 base pair bp fragment is much lower than that of the smaller 110 bp fragment DNA Fragment Analysis by Capillary Electrophoresis 31 Non specific amplification Minimizing binding to other primers 32 IMPORTANT Preferential amplification can decrease the accuracy of relative quantitation measurements Figure 3 Example of preferential amplification of the D5S346 marker In both the normal top panel and tumor bottom panel samples the peak height of the larger 124 bp fragment is much lower than that of the smaller 110 bp fragment 26 101 106 111 116 121 126 131 1 1800 1200 600 0 BB Lane 1 Normal 1800 1200 600 0 SR Lane 1 Tumor Polymerases require only the binding of the nucleotides at the 3 end to begin elongation If the 3 nucleotides bind strongly to random regions of the genome perhaps because of a 3 G C any template sequences complementary to the 3 end are amplified In this case specificity is lost because the entire primer does not specifically target the genomic region of interest Self complementary sequences within the primer can lead to the formation of hairpin structures that decrease binding specificity as well as disrupt binding stability Nucleotides in the hairpin structure are not available for binding of the target sequence The available nucleotides can be thought of as forming a smaller and therefore less specif
177. more troubleshooting information see your instrument and software user guides and the documents listed in Documentation and Support on page 199 152 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Checking data quality Sizing Quality SQ The GeneMapper Software SQ PQV reflects the similarity between the fragment PQV description pattern defined by the size standard definition and the actual distribution of size standard peaks in the sample data The metric of the Sizing Quality test is a combination of several values which measure the success of the algorithms that Identify and eliminate primer peaks based on peak shape Perform size matching ratio matching Make a size calling curve using the sizing method specified in the analysis method for more information see GeneMapper Software sizing methods on page 100 Checking samples Review the data of the size standards that fail the SQ POV as described below For with A yellow and more information see Sizing or Size Quality SQ troubleshooting on page 182 red SQ samples 1 inthe Samples tab of the GeneMapper window click amp Analysis Low Quality to Top to sort the data so that the samples that produced errors appear at the top of the table 2 In the Samples tab select the rows for the sample s that display A Check or Fail in the SQ column 3 Click L Analysis gt Size Match Editor to view the sizing informat
178. n recommendations 132 DNA Fragment Analysis by Capillary Electrophoresis instrument and consumable recommendations 131 overview 131 principle 131 troubleshooting agarose gel running 157 ambient temperature 160 autosampler alignment 161 baseline 178 buffer strength 160 capillary array 160 capillary electrophoresis 180 CEPH 1347 02 Control DNA 157 consumable issues 159 data quality 153 EPT trace 180 error message 187 extra peaks 172 GeneMapper Software 187 instrument issues 160 180 isolating the problem controls 156 matrix 161 microsatellite analysis 113 migration 168 multiplexing 29 noise 178 PCR 176 peak morphology 169 preamplification gel 190 process control 157 reagent issues 159 resolution 169 sample issues 158 signal intensity 164 size standard 182 sizing and sizing quality 182 stutter 113 temperature and humidity 161 workflow 152 Tth DNA polymerase 24 V Veriti 384 Well Thermal Cycler 58 Veriti 96 Well Thermal Cycler 58 VNTR 109 W work area DNA Fragment Analysis by Capillary Electrophoresis amplified DNA 65 PCRsetup 64 X XL PCR thermal cycler parameters 63 217
179. n page 199 DNA Fragment Analysis by Capillary Electrophoresis Overview of run modules Using controls Chapter 4 Optimizing Capillary Electrophoresis A run module contains electrophoresis parameters such as oven temperature detector cell temperature ramp rate injection time and voltage and run time or run voltage Each run module provided with the Data Collection Software has been optimized for a specific instrument polymer capillary and sample configuration run modules for the 3500 Series instrument are embedded in the instrument protocols Some settings such as injection time voltage or run time may need to be optimized for your instrument and application Updated versions of run modules for your instrument may be available on our web site To simplify troubleshooting run controls with every run for multicapillary instruments or each set of runs on 310 instruments 3500 Series instruments Run modules The 36 cm capillary arrays and POP 4 polymer are used for HID applications only Table 11 3500 Series instrument run modules Configuration 23 hours Throughputt Performance Run modules type and Sizing Precision Capillary Run t rum modules name length Polymer Time 3500 3500xL Range 50 bp 401 bp 601 bp 400 bp 600 bp 1200 bp Fragment analysis 50 cm POP 7 lt 40 min 2280 2840 lt 40 to 0 15 0 30 NATT FragmentAnalysiss0_POP7 zd F
180. n relate product yield and specificity as well as enzyme fidelity to the free magnesium ion Mg concentration free Mg total Mg total dNTP 2 EDTA In general an increase in free magnesium concentration leads to an increase in product yield but a decrease in specificity and fidelity To identify the magnesium concentration that gives the best compromise between yield and specificity in the presence of 800 uM total dNTP concentration run a MgCl reaction series in 50 uM increments over the range from 100 to 400 uM MgCl The concentration of template in a sample can affect the success of PCR amplification Too much template promotes non specific binding of primers to secondary sites or changes the pH of the reaction mix Too little template can result in poor yields especially if the template is degraded Even very low template concentrations 10 copies are often sufficient for successful PCR amplification If the starting sample is DNA you can use up to 20 000 copies of the target to start optimization experiments In general this translates to e 1to5ng of cloned template e 200 pg to 1 ng of genomic DNA Start optimization experiments with less genomic DNA if starting sample is limited With clean good quality genomic DNA 500 to 1000 pg of starting sample is typically sufficient DNA Fragment Analysis by Capillary Electrophoresis 59 Enzyme concentration For most PCR applications 2 0 to 2 5 units of AmpliT
181. nalysis by Capillary Electrophoresis Troubleshooting m Troubleshooting workflow 0 A 152 Refer to the following sections for troubleshooting solutions and information on how each component of the system can affect data R Checking data quality 6 eee eh 153 m Running controls to isolate a problem 156 Ie a SIT 158 m Reagent and consumable issues eens 159 E Instrument and ambient condition issues 0 cece ee 160 Refer to the following sections for symptom troubleshooting information m Symptoms you may Observe K 162 B Irregular signal intensity troubleshooting 00 0 eee eee eee 164 S Migration troubleshooting 0 0 c cece eee eee 168 B Abnormal peak morphology troubleshooting 5 6060 cece eee 169 m Extra peaks troubleshooting 0 0 c cee eee eens 172 m PCR troubleshooting nss miete n aia II 176 m Irregular baseline troubleshooting 6 00 c cece es 178 m Instrumentation troubleshooting 66 66 c eee ee eens 180 E Sizing or Size Quality SQ troubleshooting 0 0 182 E GeneMapper Software troubleshooting s s s sss cece cece eee ee nes 187 S Preamplification gel troubleshooting 0 0 6 c occ ee eens 190 Refer to the following sections for procedures to solve issues W Desalung gs SS das ca Grae Rie Sh sess Rege sung Nas Ie EI PEE EIS 190 m Evaluating 310 Genetic Analyzer multicomponent matrix quality
182. nalysis by Capillary Electrophoresis 11 Contents 12 Ordering Information osse exe See e e e e waits incase cem Eres ese ks 193 Thermal cyclers and accessories 000 e eee eee eee arnee 193 Genetic analyzers and consumables 000cc cece eee ees 194 GeneScan size standards 2 0 0 cece cnn tenet eden nent eens 197 Matrix standards for spectral calibration cece eee eee eae 197 Installation standards 0 0 0 rn 197 Reagent d eme Ro LR oth Gy aan ORE e Gale beards eade Sie e ER 198 Other user supplied materials 198 Documentation and Support e e e e cece teens 199 Instrument documentation cece n 199 GeneMapper Software documentation cece c eee e ete e 199 Peak Scanner Software documentation cece eee ence eee eens 200 Application documentation xs e x K e K e eee ene ence le 200 Obtain SDSS ci eR e T ve Eel tI es e ue Mee HG eee ee we IF E TY 201 Obtaltt SUppOFEbss uon on etos tee oe bo ees NL MeL Ubi d Let a 25 201 RETEREN COS A eT 203 EGLI cw E 207 GOK NE C EMI DOLES 211 DNA Fragment Analysis by Capillary Electrophoresis About This Guide IMPORTANT Before using the products described in this guide read and understand the information the Safety appendix in the documents provided with each product Revision history Revision Date Description A August 2012 New document Purpose This guide is intended for custo
183. nces in prion genes Prion diseases are caused by abnormally folded isoforms of host encoded proteins Use the SNaPshot Multiplex System to screen for SNPs in the genes that code for these proteins For instance polymorphisms at codons 136 154 and 171 of the PrP gene in sheep and goats can lead to abnormally folded isoforms of the protein product to result in scrapie e Thermal cycler Veriti or GeneAmp 9700 for fast thermal cyclers use a 1 C second ramp rate 2720 Genetic analyzer 3500 Series 3730 Series 3130 Series or 310 instruments Polymer and capillary array see Run modules on page 69 for the polymer and capillary array length combinations supported on each instrument e GeneScan 120 LIZ Size Standard e DS 02 dye set IMPORTANT Throughout a set of experiments use all the same equipment run parameters polymers dyes and so on Consistent conditions are required to avoid mobility shifts that interfere with accurate interpretation of data DNA Fragment Analysis by Capillary Electrophoresis Chapter 7 Single Nucleotide Polymorphism SNP Genotyping Experiment and This is a Life Technologies supported protocol primer design recommendations Workflow 1 Minimum primer length is 23 nt however it is strongly recommended that primers shorter than 36 nt be tested before multiplexing HPLC purification of primers longer than 30 nt is recommended Heterogeneous primer populations will lead to m
184. nd concentration DNA Fragment Analysis by Capillary Electrophoresis 87 88 Virtual Filter Set C Virtual Filter Set C dye set DS 34 is used on 310 instruments The emission maximum of 6 FAM dye the recommended blue dye for the Virtual Filter Set C is very close to the laser wavelength of 514 5 nm Thus the window for collected blue light intensity data is offset to longer wavelengths and does not contain the emission maximum of 6 FAM dye The emission maximum of 6 FAM dye is also very close to the detection region for the green TET dye Matrix files made for Virtual Filter Set C are especially susceptible to minor changes in run conditions If you are using Virtual Filter Set C watch for evidence of matrix problems and create a new matrix as soon as problems appear Factors affecting matrix quality Aging reagents e Buffer type and concentration Polymer type Denaturing or non denaturing conditions e Run temperature DNA Fragment Analysis by Capillary Electrophoresis Data Analysis with GeneMapper Software and Peak Scanner Software la OVERVIEW uos art a ae ele Ale eae A ead ee p een Ves Ee 89 E GeneMapper Software features 0 0 0 ccc eee cece cece ee 91 E Peak Scanner Software Features 0c cece cece eee es 92 R WorkllOw iu gah cca aed C RTE Latte gee booed re Fee e es 93 m GeneMapper Software peak detection settings 94 m GeneMapper Software peak start and end settings
185. nd non denaturing applications t For non denaturing applications only Matrix standards for spectral calibration Dye sets have been tested and optimized for the instrument except where noted Dye Set part numbers Instruments DS 02 DS 20 DS 30 DS 31 DS 32 DS 33 DS 34 3500 Series 4323014 Not 4345827 4345829t 4345831 4345833 Not tested tested 3730 Series 43230144 Not 43458271 4345829t 43458317 4345833 Not tested tested 3130 Series 4323014 Not 434445827 4345829 4345831 4345833 Not tested tested 310 4323050 401114 401546 401546 4312131 4318159 401546 402996 402996 4313939 t Dye primer matrix standards T We have tested this dye set but have not optimized for this instrument Installation standards Installation standard Source For use with DS 33 GeneScan Installation Standards 4376911 GeneScan 600 LIZ Size Standard v2 0 6 FAM VIC NED and PET dyes DS 33 GeneScan Installation Standards 4330397 GeneScan 500 LIZ Size Standard 6 FAM VIC NED and PET dyes DS 30 GeneScan Installation Standards 4316144 GeneScan 500 ROX 6 FAM HEX and NED dyes DNA Fragment Analysis by Capillary Electrophoresis 197 Reagent kits Item Source AFLP Kits AFLP Selective Primer Kit 4303050 AFLP II Selective Primer Kit 4303051 AFLP EcoRI Ligation Amplification Module 402941 AFLP Msel Ligation Ampli
186. ne Amplification of non specific products during PCR See PCR troubleshooting on page 176 Degraded or incorrectly stored Hi Di Formamide can cause low signal and degraded products Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 Capillary is contaminated Perform a water wash Weak or low signals and or an elevated baseline See Irregular signal intensity troubleshooting on page 164 High salt concentration Salt preferentially injects smaller fragments and inhibits injection of larger fragments so the majority of salt may have been injected in the first injection Re inject the sample If signal intensity does not increase see Desalting on page 190 Electrical noise Adequate signal strength with noisy data Secondary hybridization site is present on primer which results in many extra peaks Contact Life Technologies Evaluate primer design Impure primer You may see a shadow sequence of N 1 HPLC purify the primer DNA Fragment Analysis by Capillary Electrophoresis 179 Instrumentation troubleshooting Some data quality issues are not sample related but are caused by settings or conditions used for the instrument run Note The color of the current trace varies between versions of Data Collection Software Symptom Possible Cause Action Low or fluctuating current Expired o
187. ng 00 e e e eens 151 Troubleshooting workflow ssssssssssssssslsssss I I I ln 152 Checking data quality pe eec TL e ER exe ee tte 153 Sizing Quality SQ PQV description 00 cece cece eee e teen I 153 Checking samples with yellow and red SQ samples ccc cece cence eee 153 Examining the raw data for red SQ 1 eens 154 Examine the sample info raw data and EPT trace nnan e cece cece 154 Running controls to isolate a problem cece eee eee eee eens 156 Sizestandard a ose A P inn neds re T Mins ha AN a le ld Na AER 156 Installation standard 2 eroi leas pes ure n xa ee ewes Sie 156 Agros gal serran m 157 DNA template control sores 0 a NR 0 RR R KR depi n e mh 157 Sarmpled5sues eue rb ete bd ARD ord er dt mead Site bU et tees 158 Sample concentration iiilisiulsllllsllsss n 158 Sample contamination eee eee en 158 SalEconcentration cuv od da eee ee Seige CRIME Wa eae E META 158 Reagent and consumable issues 020e cece eect ee 159 Haboratory Water ol soeur tes duit Hala trs hat tee ied m E 159 PE Rireagents o5 facta tins otro te e elec HIM Ls 159 HisDi form mlded irr EYE E Say Ak es ertet tos 159 Polym tr incu T Taa RKR e eL ERR e eS eR etna en ieee Eee eI qu 159 Size Standard i cere seur Re OR whee c aue nib mem e mee ERU Yu nce dod e Ye 160 lonic buffer strength not applicable to 3500 Series Instruments 000 ccc ccc essen 160 DNA Fragment Analysis by Capil
188. ng efforts positional cloning comparative genomics and to determine the size and structure of genomes The SNaPshot Multiplex Kit Luo et al provides an effective easy and cost effective solution for high throughput BAC fingerprinting DNA Fragment Analysis by Capillary Electrophoresis Chapter 8 Fingerprinting 8 Principle of the In BAC fingerprinting analysis using the SNaPshot Multiplex Kit BAC clones are analysis subjected to restriction endonuclease to generate fragments of various lengths that end in A C G or T The SNaPshot chemistry then labels the fragments with the corresponding bases by single base extension to create a distinct DNA fragment pattern or fingerprint for each clone The clones are then mapped based on the order of the overlapping parts of fingerprints with other clones of the same genome Figure 34 SNaPshot restriction fragment labeling Recessed strand 5 3 Label template NNNNNNTCGGACCATGACTGATTGC NNNNNNAGCCTGGTACTGACTAACGCEGGNN SNaPshot rela t E reaction MIX ddUTP ddATP NNNNNNTCGGACCATGACTGATTGCG NNNNNNAGCCTGGTACTGACTAACGCCGGNN Electrophoresis ae or Y T Table 19 Example of possible six base cutters for restriction endonucleases and dyes used in the SNaPshot Multiplex Kit Restriction Restriction Fluorescent Restriction fragment ddNTP endonuclease site dye color EcoRI GAATTC A dR6G Green BamHI G GATCC G dR110 Blue Xbal T
189. nologies supplies the GeneAmp PCR Carryover Prevention Kit information Part no N808 0068 and AmpErase UNG Part no N808 0096 to ensure that PCR products are not reamplified in subsequent PCR amplifications 66 DNA Fragment Analysis by Capillary Electrophoresis Optimizing Capillary Electrophoresis B Safety informations eck Ee er RU ERE Hate ad eke pies ogres ges 68 L e n ocius ub b ie sa Ub NA ta eo eon eiu beet 68 B 3500 Series instruments leet heehee eie d dura tarde eee m ec 69 E 3730Seriesinstruments lsssseeeesee enhn 73 W 3130 Series instruments 6 enhn 75 B l0nstruments oo tet e Satta ESL SWEAT 76 E Optimizing sample loading concentration 6 6 cece eee eens 76 B Optimizing signal intensity 0 c ccc eens 77 B Optimizing electrokinetic injection parameters 0000 cece eens 78 E Optimizing electrophoresis conditions 80 m Other factors that affect electrophoresis 0 00 rnnr trernen 82 m Understanding spatial calibration e eens 84 m Understanding spectral calibration 84 DNA Fragment Analysis by Capillary Electrophoresis 67 Safety information Overview Life Technologies Genetic Analyzers For safety and biohazard guidelines refer to the Safety section in the user guide for your instrument For every chemical read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note For the SDSs of
190. not necessary to define new size standard definitions to accommodate migration shifts DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software Determines the expected peak spacing and height ratios Uses the list of sizes from the Size Standard definition Note The values used are for example only and do not reflect typical size standard values Base 50 400 200 400 pair Evaluates peaks in the size standard data Ignores peaks that do not meet expected pattern dotted peak Base Base 50 100 200 400 50 100 200 400 pair pair less than 4x Data Data point 100 200 400 800 point 100 200 400 800 Plots the sizing curve Uses peaks that meet expected pattern 500 4 400 o S 300 P i a d Z 200 Rm ud d 100 o 6 0 O 100 200 300 400 500 600 700 800 900 Data point DNA Fragment Analysis by Capillary Electrophoresis 99 Step 2 Sizing curve and sizing Factors that affect sizing To generate the size calling curve the software plots the actual data points of the size standard against the expected size of each size standard peak The size calling method determines how the size calling curve is generated and used to size each sample The sizing method size standard definition or size standard used to generate the sizing curve e Well to read or time to read differences Electrophoresis conditions su
191. nt Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting 4 Open the new size standard definition and add or remove peaks as needed W Size Standard Editor Edit Size Standard Description Name Description GS600LIZ Factory Provided Size Standard Dye Orange Size Standard Table Size in Basepairs 400 N s 60 0 f4 80 0 ls 100 0 je 114 0 7 120 0 lg 140 0 s 160 0 fo 180 0 ii 200 0 2 214 0 13 220 0 Cae Cie J v Troubleshooting Symptom SQ A or and Possible Cause Action No sample plot is displayed for a sample with the error No sizing data A plot is not displayed if sizing fails See Sizing or Size Quality SQ troubleshooting on page 182 Note You can view data for failed sizing in the Raw view see Examine the sample info raw data and EPT trace on page 154 Size Match Editor does not display peak data Incorrect Size Standard Dye specified in size standard definition Missing size standard peaks The fragment sizes of the size standard definition do not match the positions of the detected peaks Verify that the correct dye and fragment sizes are specified in the Size Standard definition Select modify or create correct size standard definition as needed see Modifying the size standard definition on page 182 The Peak Amplitude Thresh
192. ntific literature for a specific marker or cross species marker or by following a microsatellite development protocol Fleischer and Lowe 1995 Kandpal et al 1994 The discovery and random naming of new microsatellite markers across different organisms at multiple institutions has led to inconsistent nomenclatures for microsatellites For more information on nomenclature standards for specific genomes go to the nomenclature website of the institution a few of which are listed below Human https iris ucl ac uk Rat mouse http rgd mcw edu Fly http flybase org Design primers so the range of amplicon lengths across markers in the study spans is within the size standard fragment range with two size standard peaks preceding the smallest fragment of interest and two size standard peaks following the largest fragment of interest Use 5 end labeled primers The success of microsatellite analysis depends upon the ability to detect small mobility differences The reproducible sizing and sharp peaks obtained when using the 5 end labeling method are crucial to the success of this application If you plan to multiplex design primers with similar annealing temperatures 60 C Use reverse primer tailing on one primer in each set of primers to help differentiate between peaks made by the forward and reverse DNA strands and to promote A addition See Tail on page 34 for more information DNA Fragment Analysis by Capilla
193. ntrols to isolate a problem on page 156 Note Sample contamination can mimic a degraded capillary You can determine if the capillary issue is caused by sample contamination by running clean DNA samples or the size standard alone as a control Bubbles in the capillaries Arcing Large bubbles can affect all or many of the samples in a run Large bubbles in the pump or blocks can affect the current and can cause the following No current when voltage is applied the flow of ions is blocked by the bubble A run stops during initialization if the instrument detects unstable current Leak detected error message as the air bubble compresses when the plunger moves down to fill the array DNA Fragment Analysis by Capillary Electrophoresis Pump small bubbles Pump polymer leaks Autosampler misalignment Temperature humidity Matrix spectral Issues Chapter 11 Troubleshooting e Arcing Early loss of resolution on certain capillaries If large bubbles are present you can usually see them in the upper polymer block near the array end Refer to the instrument user guide for information on removing bubbles Small micro bubbles usually only affect single capillaries Micro bubbles in the polymer path can cause Current fluctuations or no current e Decreased resolution Possible causes of small bubbles in the pump Polymer is Notatroom temperature Allow polymer to equilibrate to room temperature and pres
194. o to www lifetechnologies com Instrument documentation necument Publication number Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 3500 Series Software v1 0 4401661 Applied Biosystems 3500 3500xL Specification Sheet 106SP11 01 Applied Biosystems 3730 DNA Analyzer Getting Started Guide 4359476 Applied Biosystems 3730 3730xl DNA Analyzer Specification Sheet 106SP08 01 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Specification Sheet 106SP07 03 310 Genetic Analyzer User Guide 4317588 310 Genetic Analyzer Specification Sheet 903565 Veriti Thermal Cycler User Guide 4375799 GeneAmp9 PCR System 9700 User Guide 4303481 GeneMapper Software documentation Document Publication number GeneMapper Software Getting Started Guide AFLP9 System Analysis 440820 GeneMapper Software Getting Started Guide Loss of Heterozygosity LOH Analysis 4403621 GeneMapper Software Getting Started Guide Microsatellite Analysis 4403672 GeneMapper Software Getting Started Guide SNaPshot Kit Analysis 4403618 GeneMapper Software Reference and Troubleshooting Guide 4403614 GeneMapper Software Installation and Administration Guide v4 1 4403614 GeneMapper Software Reference and Troubleshooting Guide v4 1 4403673 GeneMapper Software Quick Reference Guide v4 1 4403615 DNA Fragment Analysis by Capillary Electrophor
195. ogy changes for example peaks become broader are tailing or are below 50 RFU then the problem may be in the instrument reagents or Hi Di Formamide Ifthe peak profiles for the size standard alone are sharp and very well defined add your product to the same wells and re run Ifthe peak morphology then changes for example peaks become broader show tailing are less than 50 RFU then contamination may be contributing to the problem Note The size standard peak heights are affected by the presence of sample because the sample introduces salt and competes for entry into the capillary during injection If the size quality fails in the presence of sample it indicates a problem with the PCR product for example it may contain too much salt Installation Installation standards contain pooled PCR products amplified from microsatellite loci standard present in CEPH individual 1347 156 DNA Fragment Analysis by Capillary Electrophoresis Agarose gel DNA template control Chapter 11 Troubleshooting To run the installation standard e For all platforms you can load the installation standard as a regular run and view the results in the GeneMapper Software For instructions refer to the appropriate instrument user guide Alternatively for a 3500 Series instrument you can run a performance check which produces a report quantifying each peak refer to your instrument user guide Life Technologies currently supplies
196. ohesive end ligation units If the problem persists repeat the restriction ligation with fresh enzymes and buffer Use an agarose gel to check the reaction results Refer to the AFLP Plant and Microbial Protocols for more information see AFLP applications on page 200 TEO 1 is buffer not properly made or contains too much EDTA Add the appropriate amount of MgCl solution to amplified reaction Remake the TEO 1 buffer Insufficient enzyme activity Repeat the experiment with the recommended amount of restriction enzyme ligase and AmpliTaq DNA Polymerase Note 1 Weiss unit 67 cohesive end ligation units Hairpin secondary structures in PCR primers Primer design See Primer design guidelines on page 29 DNA Fragment Analysis by Capillary Electrophoresis 177 Symptom Possible Cause Action Primer dimer formation e MgCU2 concentration e Annealing temperature in the PCR See Optimizing PCR on page 55 Primer design See Primer design guidelines on page 29 Too much primer added to reaction Prepare new reaction Primer over amplification due to insufficient or poor quality template Prepare new reaction Irregular baseline troubleshooting Excessive noise or an elevated baseline affects both sizing and genotyping results Symptom Possible Cause Action Constant elevated signal in raw data e W
197. old of the dye color associated with the size standard is set too high or low in the analysis method Adjust the analysis method so that the peak detection threshold associated is greater than the height of the noise signal See GeneMapper Software peak detection settings on page 94 IMPORTANT We do not recommend decreasing the threshold below 50 RFU Expired or degraded size standard Use fresh size standard DNA Fragment Analysis by Capillary Electrophoresis 183 Symptom SQ A or and Possible Cause Action Missing size standard peaks continued Incorrect concentration of size standard in sample loading reagent Increase the concentration of size standard added to subsequent runs Size standard peaks are not migrating as expected during a normal run time See Migration troubleshooting on page 168 Incorrect injection settings for example the injection time is too short Review the injection settings of the run module for errors High salt concentration Salt preferentially injects smaller fragments and inhibits injection of larger fragments so the majority of salt may have been injected in the first injection Re inject the sample If signal intensity does not increase see Desalting on page 190 Smaller size standard fragments are not labeled Size standard peak and primer peak are in the same read region see figure be
198. om the allele peak In addition the number of allele peaks depends on whether the individual tested is a heterozygote or homozygote Dinucleotide repeats in a homozygous individual The GeneMapper Software electropherogram of a dinucleotide repeat marker from a homozygous individual 190 bp 190 bp is shown in the following figure Figure 24 Stutter peaks in a dinucleotide repeat electropherogram homozygote a Stutter Bands m i so Y Xl 0 Typical pattern for a dinucleotide repeat marker from a homozygous individual DNA Fragment Analysis by Capillary Electrophoresis 115 The peaks at 188 bp 186 bp and 184 bp show the typical 2 bp stutter pattern seen with dinucleotide repeats They represent the 2 bp 4 bp and 6 bp stutter peaks from the true 190 bp true allele peak Dinucleotide repeats in a heterozygous individual 8 bp The GeneMapper Software electropherogram of a dinucleotide repeat marker from a heterozygous individual 139 bp 147 bp is shown in the following figure Allele sizes differ by 8 bp The 2 bp stutter peak to the left of each allele peak is always of lower intensity than the allele peak itself The larger 147 bp allele peak is of lower intensity than the smaller 139 bp allele In heterozygotes the higher molecular weight allele that is the allele peak further to the right in electropherograms often produces a fluorescence signal of lower inten
199. oo high the instrument cannot measure the true value of the signal and consequently cannot compensate for the spectral overlap between the dyes As a result artifact peaks called bleed through or pull up peaks can appear beneath the sample peaks These pull up peaks can corrupt both automated sizing because extra peaks are present in the size standard dye color and the analysis of samples because the size standard is present in each sample If signal intensity is high you can Dilute the template before PCR Dilute the amplified sample before adding to formamide Decrease the sample injection time and or injection voltage When signal is too low the signal to noise ratio is also low and makes it difficult to discriminate between sample peaks and common background fluctuations If signal intensity is low you can Increase the sample injection time or injection voltage Increase the volume of template added to the PCR reaction To minimize signal intensity variations consider the ionic strength of samples and consumables The amount of DNA injected is inversely proportional to the ionic strength of the solution Note the following Samples high in salt result in poor injections PCR reactions vary in efficiency therefore some reactions may result in higher ionic concentration post amplification e Conductivity of the solvent used for injection affects the sample injection and can cause variation in peak height
200. or more of the following Ensure that you used fresh properly prepared and vortexed matrix standard Old improperly prepared or insufficiently vortexed matrix standard can cause low signal intensity Check instrument status for any run errors Verify the correct run module was used Correct as needed and repeat the run Check the freshness and preparation of reagents e Check for possible contamination of matrix standards Make sure that there are no bubbles in the sample wells e Verify that all peaks were detected A slow running system can partially or completely cut off the blue peak Increase the run time instruments other than the 3500 series or change reagents if needed and repeat the run For troubleshooting the spectral calibration refer to the instrument user guide for your instrument Purpose of a matrix The most intense fluorescence emitted by a fluorescently labeled dye falls within a small wavelength detection range However some fluorescence emission in the detection ranges of the other dyes will always occur You create a matrix that compensates for overlap by subtracting out in the detection range of each dye the portion of the signal due to fluorescence from other dyes Run each relevant dye matrix standard separately to determine the proportional amount of fluorescence that is emitted in all dye detection regions Always create a new matrix if run conditions change such as the pH or polymer type a
201. oting on page 176 Incomplete 3 A nucleotide addition See Incomplete 3 A nucleotide addition on page 33 e Stutter See the next section See Chapter 11 Troubleshooting on page 151 for more information on stutter peaks and plus A products Identifying stutter products in microsatellite analysis Overview During the PCR amplification of di tri and tetranucleotide microsatellite loci minor products that are 1 to 4 repeat units shorter than the main allele are produced The minor product peaks are referred to as stutter peaks Stutter peaks may be caused by polymerase slippage during elongation Hauge and Litt 2003 Murray and Lai 2003 DNA Fragment Analysis by Capillary Electrophoresis 113 Estimating the amount of stutter 114 Stutter peaks appear as multiple lower peaks that precede the true allele peak These stutter peaks differ in size from the true allele peak by multiples of the length of the repeat unit The number of peaks and their intensities are proportional to the length of the repeat and the number of repeats in the PCR product Shinde et al 1993 Shorter repeat units di or tri for ex generate more stutter and dinucleotide repeats tend to generate more stutter peaks than trinucleotide repeats Stutter peaks can also be caused by off scale data For more information see Evaluating data with stutter on page 117 GeneMapper Software is optimized to filter out stutter peaks You can
202. ou to use smaller reaction volumes and fewer amplification cycles during PCR e Low cost You can add FIdNTPs to any PCR You do not need to order or synthesize fluorescently labeled primers before each PCR and you can use F dNTPs with your existing primer sets 5 end labeled primer DOOD OO UO Oo 0 Oo OOo oo ELDER Labeled nucleotides LUC TUDUU Post PCR end labeling with FIdNTPs using Klenow is an alternative to labeling during PCR Iwahana et aL 1995 Inazuka et al 1996 You can also label with FIdNTPs using traditional techniques such as random priming or nick translation DNA Fragment Analysis by Capillary Electrophoresis 25 The following figure compares the results obtained using 5 end labeled primers and F dNTPs The 5 end labeled primers give better resolution but F dNTPs result in higher peaks Note also the unincorporated fluorescently labeled nucleotides in the F dNTP labeled sample Figure 1 Comparison of 5 end labeled primers top panel and F dNTP labeled primers bottom panel GS es 276 Sue LE 141 Ed rg ed LACH GC Singleplexing versus multiplexing Si ngleplexi ng Singleplexing is a PCR technique in which a single target is amplified in a reaction tube This technique uses only one primer pair in each reaction and does not require as much optimization as multiplexing However singleplexing increases the cost and time per analysis
203. ped and the GO PQV is We recommend examining all samples that produce Check or Low Quality SQ flags For information on configuring and interpreting PQVs refer to the GeneMapper Software Reference and Troubleshooting Guide v4 1 Pub no 4403673 For information on troubleshooting SQ results see Checking data quality on page 153 Figure 18 illustrates an electropherogram that meets the following criteria Peak heights are 250 RFU peaks lt 50 RFU are considered to be noise Ideal peak heights are 3500 Series instruments 2175 RFU 3730 Series 3130 Series and 310 instruments 2150 RFU Peaks are sharp with no shoulders or splits The peaks corresponding to different color dyes may not be of equal intensity but the data for the less intense colors should be clearly resolvable at higher magnification All expected peaks are detected Peaks are sized properly see Size standards on page 42 If you are genotyping samples are accurately genotyped Results are reproducible DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software Figure 18 Example of a good electropherogram If electropherograms do not meet the criteria above see Chapter 11 Troubleshooting on page 151 Examining peak To examine how GeneMapper Software has defined a peak select definitions View gt Show Peak Positions The peak positions
204. phoresis and fluorescence detection the alleles of a single marker appear as different colored peaks at roughly the same size in the electropherogram plot The size of the different allele peaks will vary slightly due to differences in molecular weight of the dyes Uses unlabeled user defined primers that are customized for your target Offers multiplexing capability up to 10 plex regardless of their positions on the chromosome or the amount of separation from neighboring SNP loci Sensitive allele frequency detection 5 Compatible with all Life Technologies genetic analyzers Automated analysis using specific GeneMapper Software data analysis module Additionally the SNaPshot kit can be used for a variety of other applications BAC fingerprinting DNA methylation Low to medium throughput linkage and association studies Single locus fragment analysis DNA Fragment Analysis by Capillary Electrophoresis 121 Instrument and consumable recommendations 122 e Screen and confirm SNPs e Screen for prion gene mutation Screen and confirm SNPs The SNaPshot Multiplex System includes a variety of SNaPshot Multiplex Kits used for SNP screening and validation Each kit offers a one tube single base extension termination reagent to label DNA fragments Screen for Prion gene mutations The single base pair sensitivity of the SNaPshot Multiplex System enables you to accurately screen samples for codon differe
205. ple again Sample or reagent is contaminated Use fresh samples and reagents DNA Fragment Analysis by Capillary Electrophoresis 169 Symptom Possible cause Action Loss of resolution continued High salt concentration Salt preferentially injects smaller fragments and inhibits injection of larger fragments so the majority of salt may have been injected in the first injection Re inject the sample If signal intensity does not increase see Desalting on page 190 Bubbles or debris in polymer path Remove bubbles or clean the polymer path Refer to the instrument user guide for information Capillary array degrading 1 Perform a water wash through the polymer delivery system Refer to the instrument user guide for information 2 Replace the capillary array 3 Runa size standard 4 f the problem is present in the size standard replace reagents then run your samples again Samples are degraded because they have been sitting in the instrument gt 24 hours Run samples as soon as possible after preparation Expired or degraded polymer Hi Di Formamide buffer or water Replace the reagent then run your samples again Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 Instrument current problem See Instrumentation troubleshooting on page 180 Use of non Life Technologies reagents 1 P
206. pleted the run s and data are visible in Data Collection Software Sample files were extracted successfully 3130 Series or 3730 Series instruments e The run folder was created and saved on the instrument computer The correct number of fsa sample files were created within the run folder 188 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible Cause Action Genotypes tab is grayed alysis View Tools Help No panel specified before analysis Specify a panel in the Project window before analysis Two peaks do not separate and are detected as one peak E The software is detecting two separate peaks as one Adjust analysis method Peak Detector settings e Polynomial degree A higher setting increases the sensitivity of the curve fitting process A lower setting decreases it Peak Window size value A lower setting increases the sensitivity of the curve fitting process A higher setting decreases it Decrease the peak window size to 13 and re analyze For more information see GeneMapper Software peak detection settings on page 94 DNA Fragment Analysis by Capillary Electrophoresis 189 Preamplification gel troubleshooting Symptom Possible Cause Action No DNA smear on agarose Incomplete digestion of Check that enzymes are not expired incubation
207. primer design Repeat the experiment with a lower initial template concentration Modify the experiment to Increase addition of A Add a final extension step of 60 minutes at 72 C Increase Mg concentration Use ABI PRIsM Tailed Primer Pairs e Remove A by enzymatic treatment T4 DNA polymerase For more information see Avoiding incomplete 3 A nucleotide addition on page 34 Many small extraneous peaks appearing next to a high intensity peak Background signal is above Minimum Peak Height value Adjust the setting in analysis method High sample concentration Extraneous peaks represent non specific DNA comigrating with main fragment peak Dilute the sample Sample concentration is too high Decrease the injection time or injection voltage See Optimizing electrokinetic injection parameters on page 78 DNA Fragment Analysis by Capillary Electrophoresis 175 PCR troubleshooting Symptom Possible Cause Action Poor priming resulting in weak signal Melting temperature is too low due to low G C content and or short primer length Secondary structure of the primer particularly at the 3 end Secondary structure of the template in the region of hybridization Evaluate primer design Insufficient FldNTPs added to PCR reaction Reamplify using more FIdNTPs or examine the efficiency of the PCR Amplified DNA concentration is lower
208. profiling CRP 136 B Inter simple sequence repeat ISSR PCR 137 Overview DNA fingerprinting is a technique that is used to identify patterns that occur in genetic markers These fingerprints are specific to particular organisms A number of techniques are available for fingerprinting DNA Fragment Analysis by Capillary Electrophoresis 125 Amplified fragment length polymorphism AFLP Analysis Principle of the analysis Figure 30 AFLP analysis Advantages 126 Amplified fragment length polymorphism AFLP is a mapping technique used to visualize polymorphisms in genomic DNA The AFLP system combines the restriction fragment length polymorphism RFLP technique and polymerase chain reaction PCR to generate a large number of amplified restriction fragments from prepared genomic DNA When separated by electrophoresis the samples yield unique band patterns that when visualized by southern blot or fluorescence based fragment analysis can be used for high resolution genotyping polymorphism detection or cladistics Savelkoul et al 1999 The AFLP procedure involves digesting genomic DNA to produce a population of restriction fragments ligation of priming sites then amplified by PCR Goel et al 2006 It is sometimes considered a variation of random amplified polymorphic DNA RAPD Restriction digestion mn S lt P E ee Se Eaa Preselective A M C Selective oo axx N pii LE TJ 1mg
209. pulations of the PCR product without adversely affecting yield Examples include 5 extensions that contain restriction sites universal primer binding sites or promoter sequences Addition of 3 A The AmpliTaq and AmpliTaq Gold DNA Polymerases like many other DNA nucleotide by Taq polymerases catalyze the addition of a single nucleotide usually an adenosine to the polymerase 3 ends of the two strands of a double stranded DNA fragment This non template complementary addition results in a denatured PCR product that is one nucleotide longer than the target sequence A PCR product containing the extra nucleotide is referred to as the plus A form Incomplete 3 A nucleotide addition Because 3 A nucleotide addition rarely goes to completion without a long extension step at the end of thermal cycling that is only a fraction of the fragments receive the extra nucleotide single base ladders often form creating peak patterns that analysis software might not interpret correctly Figure 4 The resulting allele calls can be inconsistent incorrect or missing entirely Figure 4 Split peaks resulting from incomplete 3 A nucleotide addition 166 168 170 172 174 176 178 180 182 184 186 188 300 ot Withoutextensionstep xol m ml aol wool 2004 1007 0 on 166 169 170 172 174 176 178 180 182 194 186 188 L L With extension step 5004 A DNA Fragment Analysis b
210. r incorrectly stored buffer and or polymer Use fresh buffer and polymer Bubbles in polymer Remove bubbles Refer to the instrument user guide for information Anode buffer jar 3130 Series 3730 Series or 310 instruments buffer is not above required level Fill the anode buffer jar to the required level ABC 3500 Series instruments buffer is not above required level e For opened containers Pipet buffer from the overflow chamber to the main chamber e For unopened containers Invert the ABC then tilt it slightly to make sure most of the buffer is in the larger side of the container There should be less than 1 mL of the buffer remaining in the smaller side of the container Fluctuating current Arcing caused by bubbles Remove bubbles Refer to the instrument user guide for information Current too high Decomposition of urea in the polymer Use fresh polymer Incorrect buffer formulation most likely too concentrated not applicable to 3500 Series instruments Use correctly prepared buffer Arcing to conductive surface on the instrument Ensure that the ambient temperature is 15 to 30 C and the humidity is lt 80 Check for excessive condensation on the instrument 180 DNA Fragment Analysis by Capillary Electrophoresis Chapter 11 Troubleshooting Symptom Possible Cause Action Drop off of current signal Data Collection Software EPT vi
211. r orange dye signal and are detected as size standard peaks due to over saturation of sample peak signal Spectral calibration performed with the incorrect matrix standard for the dye set Decrease sample concentration Decrease injection time Perform a spectral calibration with the correct matrix standard for the dye set see Table 18 on page 84 Spectral calibration is from a different array or has not been run within the last 3 months If the peak heights that cause the pull up peak are not near the saturation limits of the instrument repeat the spectral calibration The signal in a neighboring capillary is very strong and creating a bleed through peak Decrease the sample concentration Decrease the injection time Degraded size standard A size standard can be degraded by sitting at room temperature for gt 24 hours or using improperly stored Hi Di Formamide Use fresh size standard and fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 Wrong size standard definition was used for the analysis Degraded or incorrectly stored Hi Di Formamide Re analyze with correct size standard definition Use fresh properly stored Hi Di Formamide See Hi Di Formamide storage on page 82 DNA Fragment Analysis by Capillary Electrophoresis 185 Symptom SQ A or and Possible Cause Action Sizing failures occur in a r
212. r signal than NED TM dye Therefore to generate signals of equal intensity you must load approximately three times as much NED dye labeled fragments as 6 FAM dye labeled fragments e VICO dye emits a stronger signal and is more stable than HEX dye Use VIC dye for weak amplicons Table 6 Dye Absorption max emission max and relative intensities Dye Absorption Max Emission Max Relative Intensity not Intensity to scale 5 FAM 494 nm 530 nm 100 RFU e 6 FAM 494 nm 522 nm 100 RFU TET 521 nm 538 nm 100 RFU 310 only Z vic 538 nm 554 nm 100 RFU e JOE 528 nm 554 nm 50 RFU e HEX 535 nm 553 nm 50 RFU e LIz 638 nm 655 nm 50 RFU NED 546 nm 575 nm 40 RFU 9 TAMRA 560 nm 583 nm 25 RFU ROX 587 nm 607 nm 25 RFU i PET 558 nm 595 nm 25 RFU DNA Fragment Analysis by Capillary Electrophoresis Chapter 2 Experimental Design Points to consider When you order custom primers you specify the dyes for labeling Based on the dyes when selecting you specify you must use the appropriate dye set to perform a spectral calibration dyes for custom described in Dye sets on page 41 primers Consider the following when selecting dyes One dye is needed for the size standard red or orange Using 5 dyes provides 33 greater throughput than using 4 dyes e Use the most intense dyes for PCR products with low recovery rate from lower to higher intensity Blue
213. r your instrument to select a combination of capillary information array and polymer that provide the required resolution see Instrument documentation on page 199 72 DNA Fragment Analysis by Capillary Electrophoresis Chapter 4 Optimizing Capillary Electrophoresis 3730 Series instruments Run module and Note Because of the close proximity of capillaries on the 96 capillary 3730xl instrument we recommend using the 48 capillary 3730 instrument for fragment analysis Note Sizing precision may be lower than expected for fragments lt 50 bp run on 3730 3730xl instruments with POP 7 polymer Note If you use GeneScan 1200 LIZ Size Standard download a new run module performance and optimize it before use See Downloading 3730 instrument run modules from our website on page 48 Table 13 3730 Series instrument run module Fragment Sune 3730 Analyzer 3730xl Analyzer eee run Resolution day Samples Genotypes Samples Genotypes Homes day day day day Fragment Up to 500 bp resolution with 44 4224 84 508 2112 42 2541 Analysis 0 15 bp sizing resolution t 20 genotypes sample Dye sets and matrix standards Creating a custom dye set Refer to the specification sheet for your instrument to select a combination of capillary array and polymer that provide the required resolution see Instrument documentation on page 199 Table 18 on page 86 lists the dye sets and matrix standards
214. ragment Analysis 22_POP4 22 cm POP 49 20 min 5 760 23 040 400 bp 0 50 SNP22_POP4 22 cm POP 4 15 min 38408 15 3608 120 bp 0 50 Fragment Analysis 36 POP7 36cm POP 7 35 min 3280 13 120 500 bp 0 15 Fragment Analysis 36 POP4 36cm POP 4 45 min 2560 10 240 500 bp 0 15 HID Fragment Analysis 36cm POP 4 45 min 2560 10 240 500 bp 0 15 36_POP4 SNP36_POP4 36cm POP 4 30 min 3840 15 360 120 bp 0 15 Fragment Analysis 50 POP7 50cm POP 7 50 min 2240 8 960 500 bp 0 15 Fragment Analysis 50 POP4 50cm POP 49 65 min 1760 7 040 500 bp 0 15 Fragment Analysis 50 POP6 50cm POP 6 90 min 1280 5 120 500 bp 0 15 20 genotypes injection T Standard deviation 1 base pair bp resolution at 99 99 accuracy 8 10 genotypes injection Dye sets and matrix standards Creating a custom dye set For more information Refer to the specification sheet for your instrument to select a combination of capillary array and polymer that provide the required resolution see Instrument documentation on page 199 Table 18 on page 86 lists the dye sets and matrix standards each instrument IMPORTANT We recommend using only Life Technologies dyes Life Technologies provides calibration reagents that have been optimized for our dye sets Non Life Technologies dyes or mixed isomers of dyes have variable emission spectra and require a spectral calibration generated for the specific dyes to correct for the spectral overlap betwe
215. ragment Analysis by Capillary Electrophoresis 41 However the 3500 Series 3730 Series and 3130 Series instruments do support custom dye sets For information see Chapter 4 Optimizing Capillary Electrophoresis on page 67 Size standards Functions of a size Each unknown sample is mixed with size standard before electrophoresis and run standard together in the same capillary with the same conditions Size standards perform two functions Allow sizing of sample peaks A size curve is generated for each sample Because the sizes in bp of the size standard peaks are known the sizes of sample peaks are determined through a relative comparison of migration speeds during electrophoresis The uniform spacing of size standard fragments ensures precise sizing throughout the sizing range IMPORTANT Because the called size for a fragment can differ from its actual size compare the allele calls instead of the fragment size IMPORTANT Use the same size standard instruments and instrument conditions for all samples in a study Using different size standards instruments or instrument conditions may shift the sizing of DNA fragments Precision or reproducibility is the measure of instrument ability to generate the same size consistently for a given fragment For more information see Precise versus accurate sizing on page 90 Guidelines for consistent sizing on page 90 and How the GeneMapper Software performs siz
216. ragment analysis 50cm POP 6 lt 100 mini 2112 2336 lt 20to 0 15 030 NATT FragmentAnalysis50_POP6 2990 Long fragment analysis 50cm POP 7 lt 125 min 288 2360 lt 40to 0 15 0 330 0 45 LongFragAnalysisb0 POP7 Zop HID 36 cm POP 4 lt 35 min 2312 2936 lt 60 to lt 0 15 NAT NATT HID36 POP4 2400 HID 36 cm POP 7 x26 min 2424 21272 60 to 0 15 NAT NATT HID36 POP7 2400 SNaPshot 50 cm POP 7 lt 30 min 2376 21104 lt 40 to 0 50 NATT NATT SNaPshot50_POP7 2120 t Throughput samples day The total number of samples run in 23 hours 0 5 hour for user interaction and 0 5 hours for warm up time Resolution range The range of bases over which the resolution peak spacing interval divided by the peak width at half max in a GeneScan 600 LIZ9 or GeneScan 1200 LIZ Size Standard sample sized with a third order fit is 21 The table shows the resolution range in 29096 of samples m Sizing precision Standard deviation of sizes for one allele in the DS 33 install standard sized with the GeneScan 600 LIZ Size Standard v2 0 across multiple capillaries in the same run For one injection to pass 100 of the alleles in that injection must meet the intra run sizing precision specifications The table shows the sizing precision of 100 of alleles in 29096 of samples tt Not applicable because of the size of the fragments collected in the run DNA Fragment Analysis by Capillary Electrophore
217. randed DNA at highly specific sequences called restriction sites In PCR the nucleic acid molecule that provides the sequence to amplify For example genomic DNA can be the template for a PCR reaction that amplifies a specific region within the genome Any genes whose alleles contain different numbers of tandemly repeated oligonucleotide sequences DNA Fragment Analysis by Capillary Electrophoresis 209 210 DNA Fragment Analysis by Capillary Electrophoresis Numerics 2720 Thermal Cycler 58 3 A nucleotide addition 33 310 instrument See also capillary electrophoresis dye sets and matrix standards 86 matrix overview 87 run module updating for GeneScan LIZ Size Standard 46 safety information 68 signal intensity range 77 158 specifications 68 3130 Series instrument See also capillary electrophoresis dye sets and matrix standards 86 run module updating for GeneScan LIZ Size Standard 48 run modules downloading 48 safety information 68 signal intensity range 77 158 specifications 68 3500 Series instrument See also capillary electrophoresis dye sets and matrix standards 86 performance 70 run modules 69 safety information 68 signal intensity range 77 158 specifications 68 3730 Series instrument See also capillary electrophoresis dye sets and matrix standards 86 run module downloading 48 run module optimizing for GeneScan 600 LIZ Size Standard 48 run modules 69 safety information 68 signal intensity range 77 158 spe
218. re information Follow the recommendations for AFLP analysis See Experiment and primer design recommendations on page 127 9000 DNA PCR with extraction labeled primers Digestion with restriction enzymes a 0 c i Data Electrophoresis 35 3 analysis Bo 3 1 DNA isolation and purification PCR amplification and restriction enzyme digestion Separation and detection of the digested products via electrophoresis Analysis of data to generate the fragment profile for each sample Dv RY qe d Clustering analysis based on the profile of samples from step 4 T RFLP analysis uses the same data analysis technique as AFLP See Data analysis on page 129 For documents and publications see AFLP applications on page 200 For ordering information see Ordering Information on page 193 Bacterial Artificial Chromosome BAC fingerprinting Overview 132 BAC fingerprinting provides an efficient and cost effective method of characterizing large genomic fragment libraries for genome sequencing positional cloning and physical mapping efforts Restriction endonuclease digestion of BAC clones followed by fluorescent dye labeling can be used to generate a profile or fingerprint Overlap between fingerprints are subsequently used to assemble contiguous sequences contigs in the construction of whole genome physical maps Physical maps are important resources for genome sequenci
219. re to filter out stutter peaks and detect only true allele peaks Fore more information refer to the GeneMapper Software Getting Started Guide Microsatellite Analysis Pub no 4403672 e Amplifications with an abnormally high percent stutter can indicate mixed samples or some other problem with PCR amplification or electrophoresis DNA Fragment Analysis by Capillary Electrophoresis 117 Is stutter a real Stutter once understood does not pose a real problem for microsatellite analysis and problem can aid in allele calling by e Distinguishing true allele peaks from non specific PCR products Non specific PCR products are not associated with stutter peaks Identifying alleles that fall far outside the reported allele range The percent stutter is often specific to a particular locus You can sometimes identify alleles that fall far outside the previously reported range on the basis of percent stutter For more information See Microsatellite applications on page 200 118 DNA Fragment Analysis by Capillary Electrophoresis Single Nucleotide Polymorphism SNP Genotyping R Overview of SNP genotyping e e e e eee eee 119 m SNaPshot Multiplex System ca aE X sickest eed E RE oe awe bas 120 Overview of SNP genotyping Overview A Single Nucleotide Polymorphism SNP marker consists of a single base pair that varies in the known DNA sequence thereby creating up to four alleles or variations of the marker TCGT
220. reate a stock of primers or probe reconstitute the primer or probe in sterile 1X TE buffer 1mM Tris 0 1mM EDTA pH 8 0 or sterile nuclease free water For more information refer to Reconstituting and Diluting Primers and TaqMan Probes Pub no 4370992 Measure the primer quantity with a spectrophotometer using a primer specific absorption coefficient e 20 C to 80 C for stock solution undiluted keep concentration as high as possible e 4 C for working solution diluted appropriately up to one month Using control DNA Purpose of control DNA Guidelines for use CEPH 1347 02 Control DNA Serves as a positive control for troubleshooting PCR amplification Control DNA allows you to distinguish between problems with the sample DNA the control DNA amplifies but samples do not and problems with reagents thermal cyclers or protocols the control DNA does not amplify Allows you to monitor sizing precision Because the control DNA is not used to calculate the sizing curve you can use the sizes obtained during different capillary injections to verify that sizing precision reproducibility is within acceptable limits Allows you to correlate the fragment sizes that are obtained in different runs or on different instruments Amplify at least one control DNA sample in every PCR run Include at least one injection of amplified control DNA during every series of capillary runs Use one control injection for ever
221. rent dye colors in addition to size to distinguish between markers During data collection on our genetic analyzers the fluorescence signals are separated by diffraction grating according to wavelength and projected onto a CCD camera in a predictably spaced pattern DNA Fragment Analysis by Capillary Electrophoresis Factors that affect dye signal Chapter 2 Experimental Design Although each dye emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 6 To correct for spectral overlap the software applies a multicomponent matrix A multicomponent matrix is created when you perform a spectral calibration for a dye set using a matrix standard for more information see Dye sets on page 41 and Understanding spectral calibration on page 84 Figure 6 Emission spectra of dyes Dyes 6 FAM VIC NED PET LIZ e o c 9 o 2 E LL O o N 9 o Zz o Wavelength nm Fluorescent dyes have the following characteristics Emission spectrum The intensity of emitted light fluorescence as a function of the wavelength of the emitted light Absorption excitation spectrum The intensity of emitted light as a function of the wavelength of the exciting light Absorption excitation efficiency A measure of the probability that a dye will absorb light of a certain wavelength as a percentage of the probability of absorption
222. ries instruments oos er qu 9 hatleg seen dee eee de eee K 69 R nmmod les I vee ow Pep ener IE Fede Rai eos EA ea HEIDE ET 69 Performance ee vbt kd Merde dt ee eR ertum tbe 70 Dye sets and matrix standards 2 0000 eee eee eee eee aee 70 Creating a custom dye Sgt 0 0 eect tte eee eee eee eee 70 For more information Jede E ERE ea ee UMANE AM XE 72 3730 Series instruments 0 0 eee s en 73 Run module and performance nn cece eee eee eee eee eee e eee eaee 73 Dye sets and matrix standards ssassn 000 sse 73 Creating a custom dye seti c cc c2 ben diet dene eR EIU ped eee ve pee 73 For more information cssc ber e Gaels a A DEED Seiad eee dete te eta eae 74 3130 Series instruments mesoi renad neet nalh na tra NR K a R H Nd en 75 Run modules and performance cece eee eee eee e eee e eens 75 Dye sets and matrix standards snanar 0000 cee eee ence eee eae 75 Creating a custom dye Set dee E Doe Pl aida Aged e uM 75 Forimore Information ik LI pim e pU HS LAE e 75 3T nstr mentsz iioii e rex Re ult RP Eel ERelITH E P eETIMEleE E ee EEE EE 76 Run modules and performance sese n 76 Dye sets and matrix standards snan 000 c cee eee eee sls 76 Optimizing sample loading concentration cece eect esee 76 Optimizing signal intensity isses ee diedehs oe REEL eek gah RE LQCEENQGLOBEERDA tae 77 Optimal detection ranges n 77 Balancing size standard and sample peak intensities le
223. ry Electrophoresis 111 Workflow Data analysis 112 Based on sample DNA concentration robustness of the PCR and or peak heights observed in capillary electrophoresis determine whether you need to dilute the PCR products Dilutions can range from undiluted to 1 20 in water You can pool the diluted PCR products if desired Dye labeled PCR products must be mixed in different ratios because each dye has a slightly different fluorescence signal strength see Emission and absorption excitation wavelengths and relative intensities on page 38 To avoid inaccuracies associated with pipetting small volumes prepare a master mix of reagents Prepare sufficient master mix for at least one extra reaction volume Store the master mix in the dark at 2 to 6 C for up to 1 month or at 15 to 25 C for longer A typical reaction may include 1 uL of each PCR product and 0 5 uL of the GeneScan size standard in 8 5 uL of Hi Di formamide for denaturing applications or distilled deionized water for non denaturing applications Master mix reagents are optimized for capillary electrophoresis and differ depending on the capillary electrophoresis instrument you use Select primers and size standards a Design and order primers for a microsatellite application b Optimize amplification conditions with microsatellite markers on test DNA c Order dye labeled primers PCR Capillary electrophoresis Data analysis The G
224. s from the Life Technologies Custom Oligonucleotide Synthesis Service For information see our website Order labeled and unlabeled primer pairs for the markers of interest Testing the primers and optimizing conditions with test DNA panel Testing 52 Before using primers in an analysis test the primers and optimize sample preparation PCR and electrophoresis conditions Create a panel of test DNA samples to ensure that expected alleles are detected for each marker Use DNA samples that are representative of your overall study to capture as much allelic variation as possible CEPH Individual 1347 02 Control DNA is available from Life Technologies and can be used in your test DNA panel Test DNA panel guidelines Include 8 to 16 samples Use samples of good quality that are well quantified Use equal concentrations of DNA samples Note If optimization of signal intensity is necessary for a given sample inject the sample multiple times using a range of injection parameters DNA Fragment Analysis by Capillary Electrophoresis Optimizing conditions Chapter 2 Experimental Design Order unlabeled primers for the markers of interest and optimize amplification conditions on your DNA test panel You may need to optimize a variety of parameters including annealing temperature and variables such as magnesium concentration and primer concentration to ensure that the primers work under universal conditions Bands are visualized on
225. s of experimental conditions that make hot start impractical for high throughput applications AmpliTaq Gold DNA Polymerase is delivered in an inactive state A pre PCR heating step of 10 to 12 minutes at 95 C which can be programmed into the thermal cycling profile activates the enzyme For low template copy number amplifications step wise activation of AmpliTaq Gold DNA Polymerase or time release PCR can be useful GeneAmp Gold Allows PCR to be finished in 40 minutes Fast PCR Master Mix Platinum Multiplex Designed specifically for endpoint multiplex PCR It supports easy multiplexing with minimal PCR Master Mix optimization Amplifies up to 20 amplicons in a single reaction Amplifies products from 50 bp to 2 5 kb The performance of the Platinum Multiplex PCR Master Mix over a wide range of amplicon sizes permits the amplification of templates from 50 bp to 2 5 kb greatly enhancing workflow flexibility Coupled with its 20 plex capability and absence of primer dimers it not only provides a high throughput solution but also boasts high specificity through fewer non specific primer binding events and hence less reaction and primer waste Platinum Pfx DNA Ideal for amplification of DNA fragments for high fidelity PCR applications High fidelity is provided Polymerase by a proprietary enzyme preparation containing recombinant DNA Polymerase from Thermococcus species KOD with proofreading 3 t exonuclease activity P
226. satellite analysis 110 thermal cycler parameters 62 LOH microsatellite application 16 relative fluorescence applications 17 Loss of Heterozygosity See LOH low copy amplification enzyme 22 23 M matrix 310 instrument 191 standards for spectral calibration 86 methylation assessment 149 microbial genemapping fingerprinting application 17 microsatellite analysis advantages 108 applications 110 data analysis 112 experiment and primer design recommendations 111 instrument and consumable recommendations 111 overview 107 principle 108 214 size standard GeneScan 400 HD ROX 49 size standard GeneScan 600 LIZ 111 stutter 113 troubleshooting 113 workflow 112 microsatellite instability microsatellite application 16 146 migration troubleshooting 168 MLPA relative fluorescence applications 17 143 MLVA microsatellite application 16 multicomponent analysis 26 37 Multilocus Variant Analysis See MLVA multiplexing benefits and limitations 26 guidelines 28 pooling ratios 27 software 28 strategies 27 troubleshooting 29 N noise troubleshooting 178 normalization size standard 45 P parentage analysis 110 paternity testing 110 pathogen sub typing microsatellite application 16 PCR See also thermal cycler parameters 3 addition 33 carryover 65 contamination 64 control DNA 57 DNA template 55 general 61 62 hot start 60 62 linkage mapping 62 maximize yield of specific products 61 63 non specific amplification 32
227. sel C binding site are unlabeled You may need to optimize to identify primer combinations that generate sufficient unique marker fragments for a study For example the Plant Mapping Kits contain eight selective forward primers and eight reverse primers labeled with the 5 FAM NED and JOE dye labeled fluorophores Dye Set F The possible combinations of forward and reverse primers provides 128 possible primer combinations that have been tested across several crop genomes facilitating identification of the optimal pair s for a given organism without having to design synthesize or perform quality control tests of custom primers The Appendix of the AFLPG Plant Mapping Protocol Pub no 4303146 shows primer combinations that have been successfully used for a variety of plant species and the AFLP Microbial Fingerprinting Protocol Pub no 402977 shows primer combinations that have been successfully used for a variety of microbial organisms Note that if your organism of interest does not appear in the list you can still conduct experiments by choosing primers from the most closely related species that is available In general the strategy with AFLP analysis is to generate informative fragments or enough fragments so that individuals are distinguishable However too many fragments complicate the analysis so you must empirically determine the optimum number of fragments needed for adequate discrimination As a general rule it
228. simple sequence repeat PCR Cancer Res 61 8274 8283 Blair M W Panaud O McCouch S R 1999 Inter simple sequence repeat ISSR amplification for analysis of microsatellite motif frequency and fingerprinting in rice Oryza sativa L Theor Appl Genet 98 780 792 Canzian F Salovaara R Hemminki A et al 1996 Semiautomated Assessment of Loss of Heterozygosity and Replication Error in Tumors Cancer Res 56 3331 3337 Davis L M Glenn T C Strickland D C et al 2002 Microsatellite DNA analyses support an east west phylogeographic split of American alligator populations J Exp Zool 294 352 72 Derakshani M Lukow T and Liesack W 2001 Novel bacterial lineages at the sub division level as detected signature nucleotide targeted recovery of 16s rRNA genes from bulk soil and rice roots of flooded rice microcosms Applied Environmental Microbiology 67 2 623 631 Doyle J J Doyle J L 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue Phytochem Bull 19 11 15 Ellegren H 2004 Microsatellites simple sequences with complex evolution Nat Rev Genet 5 435 45 Eschenhagen M Schuppler M and Roske I 2003 Molecular characterization of the microbial community structure in two activated sludge systems for the advanced treatment of domestic effluents Water Res 37 13 3224 3232 Fleischer R C and Lowe S 1995 Construction and screening of microsatellite enrichedgenomic
229. sis 69 Performance Table 12 3500 Series instrument resolution largest fragment and sizing General Sizing Precision of 100 of Alleles Multirun Sizing of 100 of Alleles in in 290 of Samples 290 of Samples Largest Reso union Fragment 401 600 601 1 200 401 600 601 1 200 Range in 290 Collected in 50 400 bp b b l 50 400 bp b b of Samples 290 of P P P P Samples lt 40 to 2520 2600 0 15 0 30 NA 1 bp 2 bp NA 20 to 2550 2600 0 15 0 30 NA 1 bp 2 bp NA lt 40 to 2700 21 200 0 15 0 30 1 bp 2 bp 3 bp 60 to 2400 2420 0 15 NA NA 1 bp NA NA 60 to 2400 2420 0 15 NA NA 1 bp NA NA lt 40 to 2120 2120 0 50 NA NA 1 bp NA NA Dye sets and Table 18 on page 86 lists the dye sets and matrix standards each instrument matrix standards IMPORTANT We recommend using only Life Technologies dyes Life Technologies provides calibration reagents that have been optimized for our dye sets Creating a custom dye set Non Life Technologies dyes or mixed isomers of dyes have variable emission spectra and require a spectral calibration generated for the specific dyes to correct for the spectral overlap between the dyes You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure the dye labels do not affect PCR efficiency 1 In the DyeSet library click Create 2 Enter a dye set name 3 Select
230. sity than the lower molecular weight allele suggesting a less efficient amplification of the larger fragment This phenomenon could also be caused by preferential injection of the smaller fragments Figure 25 Stutter peaks in a dinucleotide repeat electropherogram heterozygote 8 bp Stutter bands Typical pattern for dinucleotide repeat marker from a heterozygous individual where alleles differ by 8 bp Dinucleotide repeats in a heterozygous individual 4 bp The GeneMapper Software electropherogram from a dinucleotide repeat marker of a heterozygous individual 185 bp 189 bp is shown in the following figure Allele sizes differ by 4 bp Figure 26 Stutter peaks in a dinucleotide repeat electropherogram heterozygote 4 bp Typical pattern for a dinucleotide repeat marker from a heterozygous individual where alleles differ by 4 bp 116 DNA Fragment Analysis by Capillary Electrophoresis Evaluating data with stutter Chapter 6 Microsatellite Analysis S When the difference between the allele sizes is lt 4 bp a shift occurs in the height ratio between the two allele peaks compare the two preceding figures The fluorescence signal from the 4 bp stutter of the 189 bp allele is added to the signal from the 185 bp allele Dinucleotide repeats in a heterozygous individual 2 bp The GeneMapper Software electropherogram from a dinucleotide repeat marker of a heterozygous individual 216 bp 218 bp is sho
231. size standards on page 197 Matrix Standard Kits See Matrix standards for spectral calibration on page 197 0 5 mL sample tray 5572 MicroAmp 8 tube strip 0 2 mL N8010580 MicroAmp 96 well base holds 0 2 mL reaction tubes N8010531 MicroAmp 96 well full plate cover N8010550 MicroAmp 96 well tray retainer set 403081 POP 49 Polymer 5000 uL 310 Genetic Analyzers 402838 POP 6 Polymer 5000 uL 310 Genetic Analyzer 402837 Hi Di Formamide 4311320 Running Buffer 10X 4335643 Genetic Analyzer Septa Retainer Clips for 96 tube Sample Tray 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 Septa for 0 5 mL Sample Tubes 401956 t For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 196 DNA Fragment Analysis by Capillary Electrophoresis GeneScan size standards Ordering Information LIZ dye labeled size ROX dye labeled size standards Part no standards Part no 5 dye chemistry 4 dye chemistry GeneScan 120 LIZ 4324287 GeneScan 350 ROX 401735 GeneScan 500 LIZ t 4322682 GeneScan 400HD ROX 402985 GeneScan 600 LIZ 4366589 GeneScan 500 ROX tT 401734 GeneScan 600 LIZ v2 0 4408399 GeneScan 1000 ROX t 401098 GeneScan 1200 LIZ t 4379950 t For denaturing a
232. spectral calibration 0000 cece eee eee eee nnne 87 Understanding the matrix file 310 instruments onl 00 0 ccc cece een eens 87 S CHAPTER 5 Data Analysis with GeneMapper Software and Peak Scanner Software stes cin cde cos tcen Seat sius tena astare aa 89 OVEIVIEW ties 89 How the software processes data 00 cece eee cence eee eens 89 Precise versus accurate sizing 2 cece eee eee eee eee e 90 IzelatiVe SIzIlhg senate tad Pe bed or eo hip E tt Lade tated Poet der Nate 90 Guidelines for consistent sizing 90 Autoanalysis and manual analysis GeneMapper Software only 005 91 GeneMapper Software features nuanua nrn nnne rnrn rrr n 91 Peak Scanner Software Features 00 ccc cece eee eee nent nen 92 OVENVIEW icin ta eerie aac E ac bee epu secat nace de eda 92 PG ee weeds son UELUT 92 Mi m 93 GeneMapper Software peak detection settings 0 c cece cence eene 94 Peak Amplitude Thresholds cece eee ence sss 94 jnre TTT 94 Baseline Window wx LR cc N R ERR Ta EE Cep dee ede idee RES 94 Min Peak Half Width 2 000 000 cece ccc een eee 94 Polynomial Degree and Peak Window Size parameters 00 00000 c ee eeeee eee 94 Effects of varying the Polynomial Degree nnar cece cece eens 95 Effects of Increasing the Window Size Value 0000 0c e cece cece eee eens 96 GeneMapp
233. sssslesss sse 31 DNA Fragment Analysis by Capillary Electrophoresis 3 Contents Non specific amplification 0200 c ccc ee eee e eee e eens 32 Minimizing binding to other primers 2 00 e eee e eens 32 Post amplification manipulations 2 0000 eee sese 33 Addition of 3 A nucleotide by Tag polymerase 00000 cece cece cece eee 33 B ae ee LOT em te M EE Pe t EM dU os Ed 36 Dyes and chemical forms sss e eee e teens 36 Multicomponent analysis with fluorescent dyes 000000 c cece eee eee ees 36 Factors that affect dye signal cece ee eee eee see 37 Emission and absorption excitation wavelengths and relative intensities 38 Points to consider when selecting dyes for custom primers 2 000000eeeeeeee 39 Example selecting dyes cece eee eee eee e nent e ees 39 SU ae eR Ne ere an MEL ne EN 41 Dye sets and matrix standards s s naasar cece eee eee ee eee e etna eeee 41 Creating a custom dye Sgt 0 cece cece ccc e 41 Size standards oe ie ipea n s oris Seduta bedeutet dicus bubo kf 42 Functions of a size standard see 42 Size standard peak intensity iisuussusssllllllllls en 42 Selecting a GeneScan size standard 0 0 ccc cence nnn een ere 43 Peaks not used forsizing cece ee eee ranae 43 Preparing a size standard ism A RP LL HL lee ieee 43 GeneScan 120 LIZ Size Standard 44 GeneScan 500 LIZ Size
234. stelite Tutorial fi C D65264 Bue hoo 11300 2 IR II r CD ao DNA Fragment Analysis by Capillary Electrophoresis 187 Symptom Possible Cause Action al label or alleles are not falling within bins La Na E sz 206 94 Allele is migrating at a different rate than expected See Migration troubleshooting on page 168 Bin is not defined for the allele Modify the binset to define the allele Allele not labeled Peak is detected as stutter Decrease the Stutter ratio in the analysis method If the problem persists mask the stutter peak by labeling the allele as a mono peak In the Panel Manager change the marker repeat to 1 for the marker in question Peak intensity is below the Peak Amplitude Threshold in the analysis method Adjust the Peak Amplitude Threshold in the analysis method Data not sorted by name When adding samples to a project the expected data files are not listed in the Add Samples to Project dialog box The default setting in GeneMapper Software sorts the data by status rather than sample name Data was not collected as expected To resort the data Select Edit Sort or e Shift click the column header to sort by Clicking once sorts in ascending order clicking twice sorts in descending order Ensure the following on the Data Collection Software computer e The instrument com
235. sure Crack open lid seal for at least 30 to 60 minutes before use Do not leave the lid off the polymer bottle as dust may contaminate stock causing spikes in data Newly installed Valves syringes or the array port are not screwed tightly in place Refer to the instrument user guide for information on removing bubbles Polymer leaks can cause Formation of crystals which introduce contaminants that can affect the conditions of your run If the pump is not adequately pushing polymer through the array the array can clog or become contaminated Loss of resolution Autosampler misalignment can cause consistent failures in the same wells or rows of a plate Drastic changes in room temperature and humidity can cause distinct changes in migration Incorrect matrix calibration or using different conditions to calibrate than you do to run samples can cause the following Raised baseline Negative peaks Peaks under peaks especially if the highest peak is not off scale Multiple dye colors being detected as one dye color has been observed when running 5 dye samples with a 4 dye matrix DNA Fragment Analysis by Capillary Electrophoresis 161 Symptoms you may observe Irregular signal intensity Symptom See page No signal or low signal 164 Signal intensity is too high or oversaturated 166 Ski slope peak pattern 166 Decreased s
236. sus 77 If signal intensity is above the detection range 2000 cece cece e ee eee eens 78 If signal intensity is below the detection range unarna nan ee cece ees 78 Minimizing signal intensity Variation cee eee eee eee aes 78 Optimizing electrokinetic injection parameters 0000 cece eect eens 78 De tinitlOn OfeSOlUUTOMN oi ede rere e pete eh de dd embed 79 Optimizing injection time 0 0 0 cece cect ra 79 Optimizing injection voltage an 80 Optimizing electrophoresis conditions cee cece ee 80 Optimizingiruritme bce foe teo Acum ease bL eg mias 81 Optimizing r n voltage ue bb ILU eR ERREUR e ener uenis 81 Optimizing run temperature 0 00 c cee eect ence eae 82 Other factors that affect electrophoresis cece eee eee eee 82 Laboratory temperature and humidity 2020000 eee cece eee ane 82 Salt concentration ionic strength and conductivity 02ce cece rrr 82 Hi Di Formamide storage cnc n 82 Polymer handling and characteristics 000 e cece eee eee eee ees 83 DNA Fragment Analysis by Capillary Electrophoresis Contents Understanding spatial calibration 000 eee eee eee ees 84 Understanding spectral calibration 0000 sene 84 Speetral calibrationis scsi ne ot ete eoe Sen eee REN Pee 85 Evaluating the calibration results 00 ennn nrnna 86 Q Value and Condition Number ranges 0 0 0 e cece cece ss 87 Troubleshooting
237. t of genomic DNA that can be used to identify an individual Analysis of the progeny from genetic crosses to determine the relative position of chromosomal locations A meiotic mapping technique which searches for linkage between a marker and the disease gene within several generations of affected families These studies examine the inheritance of markers in both affected and unaffected members of the family and then determine whether particular markers are physically close to the disease gene of interest Linkage mapping can be used to scan the entire genome relatively easily Inter simple sequence repeat PCR See Inter simple sequence repeat ISSR PCR on page 137 A location on a chromosome The term is sometimes used more narrowly to describe the location of a gene or genetic marker Plural loci See also Marker Any observable genetic characteristic for example gene phenotype microsatellite sequence SNP that can be used to identify a genetic location To be useful in genetic studies a marker must be present in different forms to allow researchers to distinguish between individuals One marker represents one locus and one primer pair Defined by size range of expected alleles and dye color attached to primer Highly repetitive simple sequence repeats of 2 to 7 base pairs also called short tandem repeats STRs Microsatellites fall into the broader category referred to as variable number of tandem repeats VNTRs which a
238. t start techniques The hot start technique helps to minimize the formation of primer dimers or non specific products thereby increasing specificity and sensitivity of PCR This profile specifies a pre PCR heat step for activation of AmpliTaq Gold DNA Polymerase 1 Hild 3 Tmp 35 Cycles 2 Holds 95 0 95 0 72 0 72 0 5 00 0 15 55 0 0 30 7 00 0 15 Method AmpliTaq Gold General PCR Use this profile for standard PCR 1 Hid 3 Tmp 35 Cycles 2 Holds Method General PCR Return LMS2 Use this profile with Linkage Mapping Set primers Linkage Mapping Set primers are for analysis of select microsatellite loci from the G n thon human linkage map 1 Hld 3 Tmp 10 Cycles 3 Tmp 20 Cycles S Tmp x 10 3 Tmp 20 Cycles 2 Holds 95 0 194 0 89 0 89 0 A2 00 0 15 72 0 9 15 Ee 0 72 0 f 55 0 0 30 55 0 0 30 10 00 0 15 0 15 0 15 Method LMS2 Return start Method LMS2 Time Release PCR Use this profile with AmpliTaq Gold DNA Polymerase This method minimizes the pre PCR activation step and adds a minimum of 10 additional cycles allowing for slow activation of the enzyme during cycling This provides a simple method where polymerase activity increases more slowly as product accumulates improving specificity 1 Hld 3 Tmp 40 Cycles 2 Holds Method F1 F2 62 DNA Fragment Analysis by Capillary Electrophoresis Chapter 3 Optimizing PCR Touchdown PCR Use this profile if the optimal annealin
239. te iaaii tine Apure IS the buffer conductivity is the sample conductivity r is the radius of the capillary sample Hep is the mobility of the sample molecules Hear IS the electroosmotic mobility CAUTION Mixing Hi Di Formamide with water generates formic acid Proper handling and storage of Hi Di Formamide is critical For quality results Aliquot the contents from the original bottle into one time use 1 5 mL or smaller tubes Minimize exposure to air and freeze thaw cycles IMPORTANT Do not freeze thaw more than two times Excessive freeze thaw cycles or storage at 2 to 8 C for more than 1 week causes hydrolysis into formic acid and formate Formate ions migrate preferentially into the capillary during electrokinetic injection causing a loss of signal intensity s Ensure that you do not contaminate Hi Di Formamide when setting up samples DNA Fragment Analysis by Capillary Electrophoresis Chapter 4 Optimizing Capillary Electrophoresis Store for up to 3 months at 15 to 25 C Store for up to 1 week at 2 to 8 C The figure below illustrates the variation in conductance in different quality formamide solutions Figure 16 Effect of formamide quality on conductance 2100 2400 2700 3000 3300 3600 3900 4200 4500 4800 6100 5400 5700 E 4 Lu 208 uS Effect of Formamide on Peak Resolution and Sensitivity GS500 ROX Internal Standard INN n 1samplea sm 17 tormamide 20805
240. the following installation standards for its capillary electrophoresis instruments see Installation standards on page 197 for part numbers If you use Then use Which uses these dyes GeneScan 600 LIZ Size DS 33 GeneScan 6 FAM VIC NED and Standard v2 0 Installation Standards PET dyes GeneScan 500 LIZ Size DS 33 GeneScan 6 FAM VIC NED and Standard Installation Standards PET dyes GeneScan 500 ROX DS 30 GeneScan 6 FAM HEX and NED Installation Standards dyes Run the PCR product through an agarose gel if the electropherogram shows miscellaneous unexpected peaks which may be due to unincorporated product Results from the gel will help to determine if your sample is contaminated You can use a DNA template control for example CEPH 1347 02 Control DNA useful for human target primers as a process control to ensure that sample preparation PCR and electrophoresis yield the expected results The results can help you determine whether failed reactions are caused by poor template quality problems with the control or problems with the primers 1 Run an agarose gel to separate the PCR products 2 Run control primer with control template to eliminate contaminated reagents as a possible cause 3 Run control template with your primers to eliminate your primers as a possible cause 4 Run control primers with template to eliminate template as a possib
241. thod H Imp WIS H eee n i T i Lena AK Peak baia Pak Quir Quy 00 aza ER ATP Ansys Seabee Dyas Pand f tae Red Use specified panel een FiPupe rara pand vano aeoe Ene Mowe pesce SRS B te d na rn Set Guo md 1130 0 5sege Mone Beasts ve a The rv tabels Details 0 t Nemmakistion Spe Mek Colin Taga Taa Typ ere Nane sides using bin naves Narre ee sirg teks Delete era alles Example analysis ISSR was used to compare two samples Agave shawii shawii from Rosarito and Agave shawii shawii from Border The figure below shows the distinct peak patterns of these two individuals Figure 39 Example ISSR data Pedegelm e Re Agave shawii shawii from Rosarito Agave shawii shawii from Border After analyzing the data in the GeneMapper Software genotypes were exported and evaluated using a spreadsheet program to Assess the consistency of genotyping for four replicate ISSR PCR reactions for each primer analyzed Calculate the alleles shared between the replicates Only those alleles with 10076 concordance were scored as true alleles and used in subsequent phylogenetic analyses True allele data for each individual for each primer were concatenated into a single list of binary states The binary data were then analyzed using the phylogenetic software MrBayes Huelsenbeck and Ronquist Ronquist and Huelsenbeck DNA Fragment Analysis by
242. tion Options for pooling in a PCR reaction are illustrated in the following figure the orange peaks are the size standard peaks e ada nman 1 singleplex reaction eq Q U 1 multiplex 9 reaction MW L 1 singleplex reactions If you pool samples after PCR If you amplify multiple PCR products This strategy is simpler and more flexible but pooling Optimize primers products from multiple singleplex PCR reactions often Use different dyes to label multiplex primers of increase the salt concentration in the loaded samples similar lengths which can cause unwanted downstream effects see Desalting on page 190 Primers cannot contain large regions of complementarity e f PCR product sizes overlap use different color dyes so they separate during electrophoresis see Dyes on page 36 Primers should have similar melting temperatures Tj Before performing the PCR perform a preliminary check for primer compatibility and test the pairs for successful co amplification Use a combination of dyes that can be detected using one spectral matrix one spectral calibration Optimize sample concentration to optimize signal intensity for each dye see Optimizing signal intensity on page 77 Optimize conditions for primers in singleplex reactions before using them in multiplex reactions to ensure the primers are suitable for your experiment DNA Fragment Analysis b
243. trol 157 isolating 55 purifying 56 quantifying 56 storing 56 dye sets dye components 41 matrix standards for 86 dyes chemical forms 36 emission 38 emission max 38 E electropherogram quality 104 electrophoresis See capillary electrophoresis EPT trace DNA Fragment Analysis by Capillary Electrophoresis examining 155 example of good quality 155 troubleshooting 180 error message GeneMapper Software 154 evaluating data electropherogram 104 stutter 113 excitation and emission max 38 experimental design considerations 21 F fingerprinting AFLP 125 applications 17 BAC 132 HiCEP 136 ISSR 137 T RFLP 131 fluorescent labeling 25 forensics See Human Identification fragment analysis applications 16 definition 15 workflow 19 G GeneAmp Gold Fast PCR Master Mix 23 GeneAmp PCR System 9700 384 Well Dual Autolid 58 GeneAmp PCR System 9700 Dual 384 well 58 GeneAmp PCR System 9700 Dual 96 Well 58 GeneMapper Software AFLP default method 129 AFLP genotypes 130 autoanalysis 91 error message 154 features 91 ISSR bins 139 microsatellite default method 112 peak detection settings 94 peak start and end settings 97 sizing 98 sizing methods 100 SNaPshot default method 123 troubleshooting 187 workflow 93 general PCR thermal cycler parameters 61 62 DNA Fragment Analysis by Capillary Electrophoresis GeneScan size standards See size standard genetic analyzer See 310 instrument 3130 Series instrum
244. ty to define the expected linear range in large fragment size standards where non linearity might be expected Expanded feature set for editing peaks that includes labeling merging and splitting peaks Customizable sizing table Ability to overlay sizing curves on analyzed data Ability to display and print plots in thumbnail view Lightweight software application with easy installation Ability to archive projects with sample files and associated reference data analysis methods size standards and so on for data sharing purposes DNA Fragment Analysis by Capillary Electrophoresis Workflow Chapter5 Data Analysis with GeneMapper Software and Peak Scanner Software Step GeneMapper Software Peak Scanner Software Set up a run file 1 Create a new project 2 Click to add samples to the project Import Sample files drag and drop or add files function Define analysis parameters In the Samples tab specify the analysis parameters for the samples in the project 1 In the Analysis Method column select or create an analysis method depending on the application used AFLP Microsatellite SNaPshot 2 In the Panel column select None or Project Specific Panel 3 In the Size Standard column select the appropriate size standard Choose appropriate size standard and analysis method Analyze Analyze the data by clicking the green arrow to analyze the samples in the pro
245. ults and have found ISSR preferable because of the reduced number of protocol steps required and the smaller amounts of DNA consumed e Less expensive and less time consuming than microsatellite based genotyping No need to clone and characterize microsatellites e Capillary electrophoresis delivers significantly higher resolution than traditional agarose gel electrophoresis thus increasing the amount of information obtained from each experiment ISSR has been used to investigate many plant and animal species in the following techniques Genetic fingerprinting Blair and Panaud et al 1999 Genetagging Ammiraju and Dholakia et al 2001 Detection of clonal variation Leroy and Leon 2000 e Cultivar identification Wang and Wu et al 2009 Phylogenetic analysis Gupta and Souframanien et al 2008 Detection of genomic instability Anderson and Brenner et al 2001 e Assessment of hybridization Wolfe and Xiang et al 1998 The versatility of this genotyping technique makes ISSR useful for researchers interested in diverse fields such as conservation biology and cancer research This is a Customer demonstrated protocol For information refer to ISSR applications on page 200 DNA Fragment Analysis by Capillary Electrophoresis Experiment and primer design considerations Workflow Data analysis Chapter 8 Fingerprinting 8 Cetyltrimethyl ammonium bromide CTAB gDNA isolation delivers high and
246. used for Cancer progression analysis Phylogenetic studies Genome scans for an organism where commercial marker panels are not available Population genetics studies Paternity testing Parentage analysis for selective breeding DNA Fragment Analysis by Capillary Electrophoresis Chapter 6 Microsatellite Analysis 6 Instrument and consumable recommendations This is a Life Technologies supported protocol Thermal cycler 2720 Veriti or GeneAmp 9700 Genetic analyzer 3500 Series 3130 Series or 310 instrument Polymer and capillary array see Run modules on page 69 for the polymer and capillary array length combinations supported on each instrument 600 LIZ Size Standard DS 33 G5 dye set AmpliTaq and AmpliTaq Gold DNA Polymerases are typically used for microsatellite analysis Like other DNA polymerases these polymerases may catalyze the addition of a single nucleotide usually A adenosine to the 3 ends of the two strands of a double stranded DNA fragment For more information see Addition of 3 A nucleotide by Taq polymerase on page 33 and Witmer et al 2003 IMPORTANT Throughout a set of experiments use the same equipment run parameters polymers dyes and so on Consistent conditions are required to avoid mobility shifts that interfere with accurate interpretation of data Experiment and primer design recommendations Identify the markers for your study by examining the existing scie
247. vative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and therefore the peak detector captures more peak structure in the electropherogram The peak window size sets the width in data points of the window to which the polynomial curve is fitted to data Higher peak window size values smooth out the polynomial curve which limits the structure being detected Smaller window size values allow a curve to better fit the underlying data DNA Fragment Analysis by Capillary Electrophoresis Chapter 5 Data Analysis with GeneMapper Software and Peak Scanner Software Function Polynomial Degree Value Window Size Value Increase sensitivity Higher Lower Decrease sensitivity Lower Higher Effects of varying The figure below shows peaks detected with a window size of 15 data points and a the Polynomial polynomial curve of degree 2 green 3 red and 4 black The diamonds represent a Degree detected peak using the respective polynomial curves Note that the smaller trailing peak is not detected using a degree of 2 green As the peak detection window is applied to each data point across the displayed region a polynomial curve of degree 2 could not be fitted to the underlying data to detect its structure 2000 Polynomial curve of degree 4 1500 black
248. widely available for a large number of microsatellite markers The small size of microsatellite loci improves the chance of obtaining a result particularly for samples containing very low amounts of DNA and or degraded DNA Thesmall size range of microsatellite loci makes them ideal candidates for co amplification while keeping all amplified alleles smaller than 350 base pairs Many microsatellite loci can therefore be typed from a single PCR Microsatellite alleles have discrete sizes allowing for simplified interpretation of results e PCR based tests are rapid giving results in 24 hours or less e PCR based tests are easy to standardize and automate ensuring reproducible results Microsatellite STRs typically contain 2 to 7 nucleotide repeats motifs and VNTRs contain 10 to 100 nucleotide repeating motifs distribution Figure 20 STRs compared to VNTRs Variable Number of Tandem Repeats VNTR AGTTC GCGTGA AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA AGTTCGCGTGA C Repeat sequence length 10 100 base pairs repeat Short Tandem Repeats STR AT GCC ATGCC ATGCC ATGCC ATGCC Repeat sequence length 2 9 base pairs repeat Often the length of the repeating unit correlates with its frequency within a genome For example in the human genome mononucleotide repeats are the most common form of microsatellites found and pentanucleotide and hexanucleotide repeats are the least common Ellegren 2004 However the fr
249. wn in the following figure Allele sizes differ by 2 bp Figure 27 Stutter peaks in a dinucleotide repeat electropherogram heterozygote 2bp 220 240 800 700 Stutter from 218 bp peak is added to the 216 bp peak 600 500 400 Stutter band 100 0 al a When the difference between the allele sizes is lt 2 bp the fluorescence signal from the 2 bp stutter of the larger base pair allele does not appear as a separate stutter peak It is added to the signal of the smaller base pair allele 300 200 cale rot eta a PP I Aq Olha The multipeak pattern seen with stutter peaks can complicate analysis particularly for samples with two or more alleles that are close in size For example small peaks in a position that is one repeat unit smaller than the true allele can be interpreted either asa stutter peak or as an allele in a minor component of a mixed sample The possible presence of stutter peaks makes precise quantitation especially important to allow the GeneMapper Software filtering algorithm to interpret the peak pattern accurately The percent stutter for a given allele is reproducible and does not depend on the quantity of input DNA or the number of loci amplified during multiplex PCR The relative reproducibility of percent stutter is important for a few reasons n many cases you can adjust the Peak Amplitude Threshold in the analysis method of the GeneMapper Softwa
250. xample below illustrates a good quality EPT trace The values for the trace may differ depending on the run module used but the shape of the trace should be similar to the example below Info Raw Data EPT Data 1 AmpFLSTR extentsion Sample 801 fsa 2000 L DNA Fragment Analysis by Capillary Electrophoresis EP Curent pn RunTemo pns 155 Note Trace colors differ between the Data Collection Software and the GeneMapper Software For more information see Instrumentation troubleshooting on page 180 Running controls to isolate a problem To simplify troubleshooting Life Technologies recommends that you run controls with every run for multicapillary instruments or each set of runs on 310 instruments In addition to controls included in each run you can run size standards installation standards agarose gels or DNA template controls when additional troubleshooting is required Size standard 1 Perform a run with only size standard using one of the default run modules that are provided with the software a Vortex the size standard for 1 minute b Add to each well of a plate 3500 Series 3730 Series and 3130 ater 310 instruments Series instruments e 0 5 uL of size standard e 0 5 uL of size standard e 9 5 uL of fresh Hi Di Formamide e 11 5 uL of fresh Hi Di Formamide c Run the plate 2 Examine the peak morphology Ifthe peak morphol
251. y Capillary Electrophoresis 27 Adjusting pooling ratios To ensure signal balance between dyes in a multiplexed sample adjust pooling ratios as needed The figure below shows the effect of different pooling ratios on signal balance In this example a pooling ratio of 3 1 1 yields balanced signal for the three dyes o Gi ive 1 34 ak a mu o om ay sat D Y 331 3 2 1527 BM 00 v e 02 12 1 1 3 3 A mili E a EHE 4 v50123212 GE 70 v9 022 12 BH 70 9 032 102 E iza Multiplex design software Multiplexing guidelines 28 Software applications are available to assist with the design of multiplex PCR Holleley and Geerts 2009 Compensating for overlapping fragment sizes If the sizes of different fragments overlap you can do the following to differentiate between them e Label overlapping products with different dyes Leave the following number of base pairs between the known size ranges Microsatellite applications 15 to 20 base pairs SNaPshot applications 8 to 10 base pairs Use different primer sites to alter the PCR product fragment lengths Load overlapping products in different wells or separate capillary injections runs Enzyme choice The high specificity of AmpliTaq Gold DNA Polymerase typically permits amplifying with elevated Mg concentrations for increased yield Primer quality Because reagents such as dNTPs are often limit
252. y Capillary Electrophoresis 33 Avoiding incomplete 3 A nucleotide addition Modify Considerations Thermal cycling conditions Increasing the time spent between 60 and 72 C promotes 3 A nucleotide addition Decreasing the time spent between 60 and 72 C inhibits 3 A nucleotide addition To use this method effectively determine the optimal thermal cycling conditions for each marker in each set of reaction conditions Promoting 3 A nucleotide addition has proven to be more successful than removing 3 A Residual polymerase activity at room temperature or even at 4 C is often sufficient to catalyze enough 3 A nucleotide addition to create genotyping problems Many protocols increase the final extension step to 30 to 45 minutes to promote 3 A nucleotide addition Magnesium ion concentration Increasing the magnesium ion concentration promotes 3 A nucleotide addition Decreasing the magnesium ion concentration inhibits 3 A nucleotide In general optimizing the magnesium ion concentration is best used in conjunction with other strategies If you choose to maximize 3 A nucleotide addition consider using AmpliTaq Gold DNA Polymerase at 2 5 mM MgCl Tail Brownstein et al 1996 found that adding additional nucleotides a tail to the 5 end of the reverse PCR primer either promoted or inhibited 3 A nucleotide addition to the forward labeled strand depending on the sequence of the added nucleotides Figure
253. y Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtain support For the latest services and support information for all locations go to www lifetechnologies com At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches DNA Fragment Analysis by Capillary Electrophoresis 201 202 DNA Fragment Analysis by Capillary Electrophoresis References Ammiraju J S S Dholakia B B Santra D K et al 2001 Identification of inter simple sequence repeat ISSR markers associated with seed size in wheat Theor Appl Genet 102 726 732 Andersen P S Jespersgaard C Vuust J et al 2003 Capillary electrophoresis based single strand DNA conformation analysis in high throughput mutation screening Hum Mutat 21 455 465 Anderson G R Brenner B M Swede H et al 2001 Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome wide allelotyping and inter
254. y variation in the electrophoresis parameters CEPH Individual 1347 02 Control DNA is available for human studies from Life Technologies Part no 403062 DNA Fragment Analysis by Capillary Electrophoresis 57 Reaction volumes and plate types Reaction volumes Using small amounts of template Plate types Reaction volumes for Life Technologies PCR thermal cyclers are 5 to 100 uL Although reaction tubes usually do not need to be sterilized or siliconized use autoclaved tubes when amplifying with quantities approximately 150 to 500 pg of starting DNA template Autoclaved PCR tubes are available from Life Technologies see Thermal cyclers and accessories on page 193 Table 9 Reaction plates for each thermal cycler Thermal Cycler Block Format Reaction Plate Type Networking Uses capability Veriti 96 Well 0 1 mL or 0 2 mL Standard 0 2 mL and Yes 10 to 80 uL medium high Thermal Cycler Alloy VeriFlex Fast 0 1 mL 96 well throughput HIBEKS formats VeriFlex Blocks provide better than gradient PCR optimization Veriti 384 Well 0 02 mL aluminum 384 well plate Yes 5 to 20 uL high throughput Thermal Cycler single block small sample volume Dual 96 Well 2 aluminum 96 well 0 2 mL format No 10 to 100 uL high throughput GeneAmp PCR 0 2 mL 96 well small sample volume System 9700 blocks Dual 384 Well 2 aluminum 384 well 0 02 mL format No 5 to 20 uL high throughput GeneAmp PCR 0 02 mL

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